Ultrasmall Ag + -rich nanoclusters as highly efficient nanoreservoirs for bacterial killing

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1 Nano Research Nano Res 1 DOI /s Ultrasmall Ag + -rich nanoclusters as highly efficient nanoreservoirs for bacterial killing Xun Yuan, Magdiel Inggrid Setyawati, David Tai Leong ( ), and Jianping Xie ( ) Nano Res., Just Accepted Manuscript DOI: /s on December 2, 2013 Tsinghua University Press 2013 Just Accepted This is a Just Accepted manuscript, which has been examined by the peer review process and has been accepted for publication. A Just Accepted manuscript is published online shortly after its acceptance, which is prior to technical editing and formatting and author proofing. Tsinghua University Press (TUP) provides Just Accepted as an optional and free service which allows authors to make their results available to the research community as soon as possible after acceptance. After a manuscript has been technically edited and formatted, it will be removed from the Just Accepted Web site and published as an ASAP article. Please note that technical editing may introduce minor changes to the manuscript text and/or graphics which may affect the content, and all legal disclaimers that apply to the journal pertain. In no event shall TUP be held responsible for errors or consequences arising from the use of any information contained in these Just Accepted manuscripts. To cite this manuscript please use its Digital Object Identifier (DOI ), which is identical for all formats of publication.

2 Table of Content for Ultrasmall Ag + -Rich Nanoclusters as Highly Efficient Nanoreservoirs for Bacterial Killing Xun Yuan, Magdiel Inggrid Setyawati, David Tai Leong, * and Jianping Xie * Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, , Singapore Ultrasmall thiolated Ag + -rich nanoclusters showed higher antimicrobial activities than Ag 0 -rich nanoclusters with the same size and surface ligand. Prof. J. Xie, Prof. D. T. Leong, 1

3 Nano Res DOI (automatically inserted by the publisher) Research Article Ultrasmall Ag + -rich nanoclusters as highly efficient nanoreservoirs for bacterial killing Xun Yuan, Magdiel I. Setyawati, David T. Leong ( ), and Jianping Xie ( ) Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, , Singapore X. Yuan and M. I. Setyawati contributed equally to this work. Received: day month year / Revised: day month year / Accepted: day month year (automatically inserted by the publisher) Tsinghua University Press and Springer-Verlag Berlin Heidelberg 2011 ABSTRACT Metallic silver (Ag) and its ability to combat infection have been known since ancient history. In the wake of nanotechnology advancement, silver s efficacy to fight broad spectrum bacterial infections is further improved in the form of Ag nanoparticles (NPs). Recent studies ascribed Ag NPs broad spectrum antimicrobial properties to dissociation of Ag + ions from the NPs, which may not be entirely applicable when the size of Ag NPs decreases to the sub 2 nm range [termed as Ag nanoclusters (NCs)]. In this paper we report that ultrasmall glutathione protected Ag + rich NCs (Ag + R NCs for short, with a dominant Ag + species in the NCs) have much higher antimicrobial activities towards both gram negative and gram positive bacteria than the reference NC GSH Ag 0 R NCs. They have the same size and surface ligand, but with different oxidation states of the core silver. This interesting finding supports that the undissociated Ag + R NCs armed with abundant Ag + ions on the surface are highly active in bacterial killing, which was not observed in the system of their larger counterpart Ag NPs. KEYWORDS noble metal nanoclusters, silver, antimicrobial agent, biomedicine, bacteria Metallic silver (Ag) and its ability to combat infection have been known since ancient history [1]. Herodotus accounted the use of silver container to keep water fresh for years during wartime [2]. Since then, silver has been used to treat many ailments including pneumonia, typhus, and leg ulcers [3, 4]. The wake of nanotechnology had allow much smaller and highly more efficient material to be Address correspondence to Prof. J. Xie, chexiej@nus.edu.sg; Prof. D. T. Leong, cheltwd@nus.edu.sg 2

4 synthesized and to be incorporated into wide variety of consumer products [5 10]. Recent studies ascribed Ag nanoparticles (Ag NPs) broad spectrum antimicrobial properties to dissociation of Ag + ions from the NPs [11], providing new inspirations in the design of high efficiency Ag based antimicrobial agents. Despite knowing the mechanism, direct usage of Ag + ions and their complexes as antimicrobial agents is not advisable as it may result in adverse cytotoxic effect on mammalian cells due to Ag + ions high affinity to biomolecules [6, 12, 13]. Strong interests therefore focus on the design of various Ag nanomaterial based antimicrobial agents, which could provide enough Ag + ion reservoir for bactericidal effect without endangering human cells through designed release of Ag + ions [14]. Theoretically, high efficiency Ag nanomaterial based antimicrobial agent should possess three features: ultrasmall size for high Ag atom efficiency, a high local concentration of Ag + ions assembled on the surface for enhanced antimicrobial efficacy, and appropriately timed release kinetics to avoid a catastrophic dissociation of Ag + ions from the nanomaterials and consequent cytotoxicity to mammalian cells. Here, we report a novel Ag based antimicrobial agent: ultrasmall thiolate protected Ag + rich NPs (thiolate Ag + R NPs for short) as nanoreservoirs with slow timed dissociation of Ag + ions. The dominant Ag + species were assembled on the surface of these ultrasmall NPs (< 2 nm) with thiolates as protecting ligands to prevent the fast dissociation of Ag + ions; thus fulfilling the above mentioned features of a highly efficient Ag based antimicrobial agent. To further prove our concept in controlled experiments, we synthesized reference NPs of the same size and ligand protection, Ag 0 R NPs but instead had Ag 0 species localized on the NP surface. Utilizing and comparing with these Ag 0 R NPs as reference, the as synthesized Ag + R NPs exhibited better antimicrobial activity for both gram negative and gram positive bacteria. Thus, exemplifying the importance of high local concentration of Ag + assembled on the NP surface in improving the antimicrobial activity. Our findings further encourage the novel design of ultrasmall Ag NP based antimicrobial agents and potentially promote the advances of this field. We aim to synthesize an ultrasmall Ag NP (smaller than 2 nm in size) [15 18], which is typically termed as nanoclusters (NCs) [19 24]. Recently, noble metal NCs (e.g., Au and Ag) have drawn interest owing to their unique molecular like properties (e.g., luminescence [25 29]) together with their great potential in biomedical applications such as bioimaging [30 33], biosensing [34, 35], and antimicrobial agent [36]. Here well developed glutathione (GSH) protected Ag NCs were chosen as our research model [37]. GSH is a naturally occurring tripeptide with a thiol group in the cysteine residue [38]. The GSH Ag + R NCs (Figure 1a) were synthesized by using previously developed cyclic NaBH4 reduction decomposition method with slight modification [36]. Briefly, AgNO3 and GSH were mixed together to form GSH Ag + complexes, followed by repeated NaBH4 reduction decomposition NaBH4 reduction procedures as well as a size focusing process of 20 hrs at room temperature. As a reference sample, the same sized GSH Ag 0 R NCs can be obtained through reducing GSH Ag + R NCs. As shown in Figure 1a, by introducing a controlled amount of NaBH4, GSH Ag + R NCs can be transformed to GSH Ag 0 R NCs. The as synthesized GSH Ag + R NCs has the same size with its reference GSH Ag 0 R NCs, which was confirmed by using electrospray ionization (ESI) and matrix assisted laser desorption ionization time of flight (MALDI TOF) mass spectrometry. As shown in Figure 1b (upper panel), four distinct peaks at ~m/z 1538, 1650, 1759, and 1980 were observed in the mass spectrum of the GSH Ag + R NCs. These peaks were assigned to be [Ag16(SG)9] 3, [Ag16(SG)5] 2, [Ag14(SG)12] 3, and [Ag15(SG)14] 3, 3

5 Figure 1. (a) Schematic illustration of the conversion process of GSH-Ag + -R NCs to GSH-Ag 0 -R NCs. (b) ESI mass spectra of the GSH-Ag + -R NCs (upper panel) and GSH-Ag 0 -R NCs (lower panel). (c) Optical absorption (solid lines) and photoemission (dash lines, λ ex = 489 nm) spectra of the GSH-Ag + -R NCs (red lines) and GSH-Ag 0 -R NCs (black lines). The inset shows photographs of the GSH-Ag + -R NCs (upper panel) and GSH-Ag 0 -R NCs (bottom panel) under visible (left items) and UV (right items) light illumination. (d) XPS spectra of the GSH-Ag + -R NCs (upper panel) and GSH-Ag 0 -R NCs (lower panel). respectively [see Figure S1 in the Electronic Supplementary Material (ESM) for detailed analyses]. The same testing conditions were used to obtain the size information of GSH Ag 0 R NCs. Similarly, four sets of peaks at ~m/z 1516, 1616, 1728, and 1946 were identified (lower panel in Figure 1b), which could be attributed to [Ag16(SG)9] 3, [Ag15(SG)5] 2, [Ag16(SG)11] 3, and [Ag14(SG)14] 3, respectively (see Figure S2 in the ESM for detailed analyses). ESI results revealed that GSH Ag + R NCs and GSH Ag 0 R NCs are generally in the same size distribution (Ag14 16). MALDI TOF mass spectra (Figure S3 in the ESM) of two NCs separately showed a peak at ~m/z 4500, supporting the same size range of GSH Ag + R NCs and GSH Ag 0 R NCs. Furthermore, transmission electron microscopy (TEM) images also confirmed that the GSH Ag + R NCs (Figure S4a in the ESM) have an indistinguishable size with GSH Ag 0 R NCs (Figure S4b in the ESM), and both are smaller than 1.5 nm. Despite the same shared size, GSH Ag + R NCs and GSH Ag 0 R NCs exhibited totally different optical properties (e.g., absorption and luminescence). Specifically, the GSH Ag + R NCs was brown in solution (inset of Figure 1c, upper panel, left item) and showed a distinct absorption peak at 489 nm (Figure 1c, solid red line). Moreover, an intense red luminescence was emitted by GSH Ag + R NCs under UV light illumination (inset of Figure 1c, upper panel, right item) with an emission peak at 643 nm (Figure 1c, dash red line). Upon the introduction of NaBH4 (15 μl) to an aqueous GSH Ag + R NC solution (~5 ml), the solution color changed to dark brown (inset of Figure 1c, lower panel, left item) accompanied with a blue shift in its optical absorption (459 nm, Figure 1, solid black line). Furthermore, the obtained GSH Ag 0 R NCs did not show any visible luminescence (inset of Figure 1c, lower panel, right item), which was validated by its photoemission spectrum (Figure 1c, dash black line). The dramatic changes in optical properties implied such two NCs may bear 4

6 dissimilar oxidation states [39], which was consistent with the fact that GSH Ag + R NCs was processed to GSH Ag 0 R NCs by NaBH4. X ray photoelectron spectroscopy (XPS) was then used to examine the oxidation states of Ag species in GSH Ag + R NCs and GSH Ag 0 R NCs. As shown in Figure 1d (upper panel), the binding energy of Ag 3d5/2 of GSH Ag + R NCs was centered at ~ ev. This broad peak was deconvoluted into Ag 0 and Ag + components with binding energies of and ev, respectively (dash blue and green lines), clearly suggesting the predominant species (~67.2 %) of Ag + in GSH Ag + R NCs. By contrast, the binding energy of Ag 3d5/2 of GSH Ag 0 R NCs was located at ev (Figure 1d, lower panel), close to that of the Ag 0 (367.7 ev), indicating the majority of Ag 0 species (~78.3 %) in GSH Ag 0 R NCs. The ESI mass spectra, MALDI TOF mass spectra, TEM, UV vis absorption spectra, luminescence spectra, and XPS analyses suggested that, GSH Ag + R NCs and GSH Ag 0 R NCs have the same size distribution and surface ligand but different oxidation states. Owing to the high local concentration of Ag + ions assembled on the NC surface, GSH Ag + R NCs showed superior antimicrobial activity compared with its reference GSH Ag 0 R NCs. Bacteria lawn of 4 different opportunistic bacteria including two types of gram negative bacteria Pseudomonas aeruginosa and Escherichia coli, as well as two gram positive bacteria Bacillus subtilis and Staphylococcus aureus, were selected as bacterial models to study the antimicrobial properties of the NCs. The susceptibilities of the four bacterial strains towards GSH Ag + R NCs (Figure 2a, column 3) was first determined by an agar diffusion assay where ultrapure water, GSH Ag 0 R NCs, and antibiotic ampicillin were applied as vehicle control (Figure 2a, column 1), reference sample (Figure 2a, column 2), and positive control (Figure 2a, column 4), respectively. Our results showed that GSH Ag + R NCs, ampicillin, and GSH Ag 0 R NCs are all active in inhibiting those four bacterial strains, as shown by the cleared agar zones (Figure 2a). Furthermore, the comparison in antimicrobial capabilities of GSH Ag + R NCs, ampicillin, and GSH Ag 0 R NCs was shown in Figure 2b, which clearly illustrated that GSH Ag + R NCs has the stronger antimicrobial capability than GSH Ag 0 R NCs, while it exhibits Figure 2. Agar diffusion assay (a) shows the presence of clear zone surrounding filter paper where the Ag NC samples (100 µm) were introduced. The clear zone periphery is demarcated by the dashed line. Scale bar = 1 cm. Interestingly, the GSH-Ag + -R NC treated samples produce wider zone inhibition (b and c), suggesting the GSH-Ag + -R NCs to be a more potent antimicrobial agent than the GSH-Ag 0 -R NCs. Ultrapure water was used as vehicle control and Ampicillin (50 μg/ml) was served as positive control. Data are means ± SD (n = 5). Student s t-test, p < 0.05, * denotes significant difference from vehicle control. # denotes significantly different between GSH-Ag + -R and GSH-Ag 0 -R NC treated samples. 5

7 comparable performance with the antibiotic ampicillin. The detailed widths of inhibition zone of P. aeruginosa, E. coli, B. subtilis and S. aureus treated by GSH Ag + R NCs, ampicillin, and GSH Ag 0 R NCs were summarized in Figure 2c. To further affirm the efficacy of the GSH Ag + R NCs against P. aeruginosa, E. coli, B. subtilis, and S. aureus, we treated bacterial media cultures with different concentrations of GSH Ag + R NC suspension from 6.25 to 800 μm (on the basis of Ag atoms) with GSH Ag 0 R NCs as reference, and observed the growth over a time course of 12 h (Figure 3a d). Our results showed that at concentrations of up to 400 μm, GSH Ag + R NCs are more active than the reference GSH Ag 0 R NCs in inhibiting the growth of gram negative bacteria such as P. aeruginosa (Figure 3a) and E. coli (Figure 3b). Similarly, at concentrations of < 400 μm, higher antimicrobial activities of GSH Ag + R NCs against gram positive bacteria (i.e., B. subtilis, and S. aureus) than GSH Ag 0 R NCs were observed (Figure 3c and d). However, at higher concentrations (e.g., 800 μm in cases of gram negative bacteria and 400 μm in cases of gram positive bacteria), GSH Ag + R NCs showed the same antimicrobial activity with GSH Ag 0 R NCs, which is not unexpected considering the excessive addition of both two NCs. The minimum inhibitory concentrations (MIC) of GSH Ag + R NCs, the MIC of GSH Ag + R NCs are 100, 100, 80, and 80 μm for P. aeruginosa, E. coli, S. aureus, and B. subtilis, respectively (Figure 3e). These results are much lower than what we observed for GSH Ag 0 R NCs (310 μm for P. aeruginosa, 400 μm for E. coli, 120 μm for S. aureus, and 150 μm for B. subtilis), further demonstrating semi quantitatively the superior antimicrobial activity of GSH Ag + R NCs over GSH Ag 0 R NCs. In addition, MIC analysis suggests that gram positive bacteria are more susceptible to the Ag NCs, as evidenced by lower MIC values. This susceptibility difference is possibly caused by the inherent difference in the bacteria cells morphology, whereby the gram negative bacteria possess an additional outer membrane, making them less susceptible to the Ag NCs. Moreover, gram positive cell membrane was built mostly by peptidoglycan [40], which contain an abundance of proteins as potential targets for Ag ion mediated cell wall disruption [41]. In contrast, the gram negative bacterial cell walls possess only 10% of the relative peptidoglycan content of gram positive bacterial cell walls [42], thus making them less susceptible to the Ag NCs antimicrobial action. Figure 3. Efficacy of GSH-Ag + -R NCs as an antimicrobial agent with the GSH-Ag 0 -R NCs as reference was assayed by its ability to reduce bacteria cell viability (a-d). The higher cell viability reduction could be seen following GSH-Ag + -R NC treatment, suggesting the higher potency of this sample when compared to that treated by GSH-Ag 0 -R NCs. Data are means ± SD (n = 3), Student s t-test, p < 0.05, * denotes significantly different from vehicle control. # denotes significant difference between GSH-Ag + -R and GSH-Ag 0 -R NC treated samples. Microbial inhibition concentration (MIC) was determined and summarized in (e). 6

8 There is an interesting question arising on why GSH Ag + R NCs has higher antimicrobial activity than its reference GSH Ag 0 R NCs. We deduced that GSH Ag + R NCs has dual antimicrobial functions compared with the reference GSH Ag 0 R NCs. Firstly, the intact GSH Ag + R NCs armed with abundant Ag + ions on the surface are highly active in bacterial killing probably because the high local concentration of Ag + ions on the NC surface could favorably interact with biomolecules on the bacteria cell wall (e.g., carbohydrates with abundant hydroxyl and carboxylic groups and membrane bound proteins laden with thiol groups) and consequently caused the cell membrane damage (Scheme 1, left side) [43], which can be referred to as the first round of bacterial killing. Next, during the killing process a large amount of oxygen reactive species (ROS) could be generated [36], which might accelerate the dissociation of Ag + ions from the NCs. The dissociated Ag + ions could initiate the second round of bacterial killing, further boosting the antimicrobial performance in a positive feedback cycle (Scheme 1, left side). By contrast, the GSH Ag 0 R NCs itself is more inert for bacteria, and its antimicrobial activity is mainly originated from the dissociation of Ag + ions (Scheme 1, right hand). Taken together, the GSH Ag + R NCs most likely have dual antimicrobial functions while the GSH Ag 0 R NCs only have single antimicrobial activity. However, more systematic studies are required to understand the underlying mechanisms. In summary, we have studied the antimicrobial activity of GSH Ag + R NCs towards both gram negative (i.e., P. aeruginosa, and E. coli) and gram positive (i.e., B. subtilis, and S. aureus) bacteria with the GSH Ag 0 R NCs as reference. A series of characterizations revealed that GSH Ag + R NCs has the same size and surface ligand with GSH Ag 0 R NCs but different oxidation states. Antimicrobial studies showed that GSH Ag + R NCs has much higher antimicrobial activity than the reference GSH Ag 0 R NCs regardless of the bacteria types. The dual antimicrobial function of GSH Ag + R NCs as the bacterial killing mechanism was also proposed. This study is of interest not only because it provides a high efficiency Ag based antimicrobial agent, but more importantly because it exemplifies the vital role of high local concentration of Ag + species assembled on the NC surface in improving the antimicrobial activity. We hope this study could offer some design principles for new Ag based antimicrobial agents for antibiotic resistant bacteria. Acknowledgements We thank Prof Ting Yen Peng (Chemical and Biomolecular Engineering, NUS) for his kind gift of B. subtilis, S. aureus and P. aeruginosa strains. This work is financially supported by the Ministry of Education, Singapore, under Grants R and R X. Yuan and M. I. Setyawati acknowledge the National University of Singapore for their research scholarships. Scheme 1. Schematic of possible antimicrobial mechanisms of GSH-Ag + -R NCs and GSH-Ag 0 -R NCs. Electronic Supplementary Material: Supplementary material (Chemicals and instruments used, detailed experimental procedures, and details of Figure S1 4) 7

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