Surface Ligand Chemistry of Gold Nanoclusters Determines Their

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Surface Ligand Chemistry of Gold Nanoclusters Determines Their Antimicrobial Ability Kaiyuan Zheng, Magdiel I. Setyawati, David Tai Leong, and Jianping Xie Chem. Mater., Just Accepted Manuscript • DOI: 10.1021/acs.chemmater.8b00667 • Publication Date (Web): 03 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018

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Chemistry of Materials

Surface Ligand Chemistry of Gold Nanoclusters Determines Their Antimicrobial Ability 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, Singapore ABSTRACT: Surface properties of nanoparticles (NPs) could greatly influence their biomedical efficacy. This paradigm drives many NPs-based antimicrobial agents as the common belief that a more positively charged surface would favor intimate interactions with the negatively charged bacterial cell wall, leading to a higher overall antimicrobial efficacy. Surprisingly, this study shows the opposite effect when using ultrasmall gold nanoclusters (Au NCs) as a model to investigate the effect of surface properties on their antimicrobial performance. Leveraging on the molecular properties of ultrasmall Au NCs, the surface properties of thiolate-protected Au NCs could be precisely controlled at the atomic level, generating a family of Au NCs with the same number of gold atoms but different surface properties. By tuning the type and ratio of surface ligands on Au NCs, more negatively charged Au NCs would produce more reactive oxygen species (ROS), leading to a better bacterial killing efficiency. This finding is in stark contrast to the previous paradigm and suggests the complexities of the nanomaterial-cell interactions, shedding some light on the design of high performance NPs-based antimicrobial agents.

INTRODUCTION Physicochemical properties of nanoparticles (NPs) are crucial in determining their biological fate and their subsequent applications as nanomedicine.1-10 Tailoring NPs’ physicochemical properties has been one of the main strategies adopted by the nanotechnologists to confer the NPs with the desired biomedical properties.11-17 Nanomedicine efficacy, however, hinges on their ability to initiate interaction with cells that led to their internalization. This initial interaction is greatly affected by the moieties that presented on both the NPs and the cells.6, 18-22 Positively charged moieties are incorporated on the NP surface to cater the long-held belief that these moieties would yield intimate interactions with the negatively charged bacterial cell wall.23, 24 For example, immobilized high density antimicrobial peptides (AMPs) on the gold nanoparticles (Au NPs) could achieve a wide-spectrum antimicrobial ability.25, 26 Furthermore, customized positively charged or zwitterionic Au NPs were generated to improve their initial interaction with bacteria, thus damaging the bacterial membrane and resulting in a certain antimicrobial efficacy.27, 28 After initial internalization, the surface properties of NPs directly affect the cellular response and intracellular activity, especially the cellular metabolism. It has been reported that the length of surface ligands on copper (Cu) NPs could influence their capability in generating intracellular reactive oxygen species (ROS).29 Surface ligands on Au nanoclusters (NCs) could also affect their cellular uptake and the subsequent intracellular redox signaling.30 Recently, we found that reducing the size of Au NPs down to molecular-like Au NCs (~1 nm) has conferred antimicrobial ability to the otherwise inert Au NPs.31 This antimicrobial activity stemmed from the internalization of

Au NCs and the subsequent induction of intracellular ROS generation.31 Considering the role of surface properties in regulating the NPs’ interaction with the bacterial cells and their cellular metabolism response, we hypothesized that we could further tune the Au NCs’ antimicrobial properties through their surface chemistry. One characteristic property of Au NCs is the precise control of their composition at atomic level, which could not be achieved in their larger counterparts – Au NPs with size above 3 nm.32-35 In general, the molecular-like Au NCs protected by thiolate ligands (an efficient protecting ligand that could confer good stability to Au NPs in solution as well as in biological systems) have molecular formula of Aux(SR)y, where SR represents the thiolate ligand, and x and y are the numbers of Au atoms and thiolate ligands, respectively. Advanced nanocluster chemistry can manipulate the type and composition of protecting ligands on Au NC surface, leading to various thiolate-protected Au NCs with precise size, composition and designed surface properties.36-44 Leveraging on the molecular features of Au NCs, in this study we chose Au25SR18 (thiolate-protected Au NCs with 25 Au atoms and 18 thiolate ligands) as a model antimicrobial molecule. We have precisely tuned the Au NCs’ surface properties by controlling the type as well as the composition of surface ligands, while keeping the number of Au atoms in the core unchanged.45 We demonstrated that the surface properties of Au NCs could influence their ROS generation ability, which could directly lead to different antimicrobial efficacy. The unexpected finding that more negatively charged Au NCs could show higher antimicrobial efficacy also suggests the complexities of the nanomaterial-cell interactions, shedding some light on the design of high performance NCs-based antimicrobial agents.

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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), 3-mercaptobenzoic acid (MBA), L-cysteine (Cys), cysteamine hydrochloride (Cystm), 2-mercaptoethanol (MetH), sodium borohydride (NaBH4), 2’,7’-dichlorofluorescein diacetate (DCFH-DA), lysozyme, lysostaphin, paraformaldehyde (PFA), agar and mineral oil were purchased from Sigma-Aldrich; sodium hydroxide (NaOH) was from Merck; 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 (S. aureus; ATCC 25923) was a kind gift from Prof. Tan Kai Soo (National University of Singapore, NUS). Escherichia coli (E. coli; ATCC 700926) was obtained from the American Type Culture Collection (ATCC, USA). Carbon monoxide (CO) was provided by SOXAL. Instruments Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to determine the concentration of Au atoms in solution, which was measured through iCAP 6000 Series, Thermo Scientific. UV-vis absorption spectra of the samples were recorded by a Shimadzu UV-1800 photospectrometer. The molecular formulas of Au NCs were determined by electrospray ionization (ESI) mass spectrometry on a Bruker microTOF-Q system. Transmission electron microscopy (TEM) images were taken on a JEOL JEM 2010 microscope operating at 200 kV. Hydrodynamic diameter and zeta 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 microplate reader Biotek H4FM. The bacteria fluorescent images were taken with an epifluorescence microscope Leica DMI6000. The Z-stack fluorescent images were taken with Nikon A1 confocal microscope. Synthesis of Au25 NCs Protected by Different Thiolate Ligands Au25MHA18 and Au25Cys18 NCs The synthesis of Au NCs was following a reported method.36 In a typical synthesis, freshly prepared aqueous solutions of HAuCl4 (20 mM, 0.25 mL) and MHA or Cys (5 mM, 2 mL) were mixed in water (2.35 mL), leading to the formation of white Au(I)MHA or Au(I)-Cys 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 NaBH4 in NaOH solution (0.2 M, 10 mL). Afterwards, NaBH4 solution (0.1 mL) was added into Au(I)-MHA complexes or Au(I)-Cys complexes, and the Au NCs were collected after 3 h reaction. After synthesis, a stirred ultrafiltration cell (Model 8010, Millipore Corporation, USA) with a semi-permeable membrane of 3000 Da

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molecular weight cut off (MWCO) was used to purify Au NCs in solution. Au25CystmxMHA18-x and Au25MetHxMHA18-x NCs The biligand-protected Au25 NCs were synthesized by keeping all the experimental conditions the same as the above except for the second thiolate ligands introduction process. The total volume of thiolate ligands was kept as 2 mL, and the volume of each thiolate ligands was adjusted as follows: MHA (from 1.9, 1.75, to 1.25 mL) and Cystm (from 0.1, 0.25, to 0.75 mL), MHA (from 1.9, 1.75, to 1.25 mL) and MetH (from 0.1, 0.25, to 0.75 mL). In general, the final ratios of MeHA to Cystm (or MetH) are 19/1, 7/1 and 5/3, respectively. After synthesis, a stirred ultrafiltration cell (Model 8010, Millipore Corporation, USA) with a semipermeable membrane of 3000 Da MWCO was used to purify Au NCs in solution. Au25MBA18 NCs The synthesis of Au25MBA18 NCs was following a reported method.46 In a typical synthesis, freshly prepared aqueous solutions of HAuCl4 (40 mM, 0.25 mL) and MBA (50 mM in 0.15M NaOH, 0.4 mL) were mixed in water (9.5 mL) under mild stirring to form lightyellow Au(I)-MBA complexes for 2 h. Afterwards, the pH of the solution was adjusted to 11.6, and CO was bubbled for 2 min. Then, the bottle was sealed airtight, and the reaction was allowed to proceed for 72 h. After synthesis, a stirred ultrafiltration cell (Model 8010, Millipore Corporation, USA) with a semi-permeable membrane of 3000 Da MWCO was used to purify Au NCs in solution. Bacterial related experimens All bacterial related experimental details are available in the Supporting Information.

RESULTS AND DISCUSSION Au25SR18 has a molecular structure consisting of an icosahedral Au13 core protected by six [SR-Au(I)-SR-Au(I)-SR] motifs (twelve Au atoms and eighteen thiolate ligands), as depicted in Figure 1a.47 Au25SR18 is one of the most stable Au NC species in solution, and their surface properties (ligand types and combinations) could be precisely controlled. Thiolate ligands with different functional groups could be used to protect Au25 NCs, and the composition and stoichiometric ratio of bi-ligand-protected Au25SR18 could also be precisely adjusted. The as-synthesized Au NCs have ultrasmall core size below 2 nm, as indicated in transmission electron microscopy (TEM, Figure S1a) images. Their hydrodynamic diameter is ~4 nm (Figure S1b). As our hypothesis hinges on the different surface properties of the antimicrobial Au NCs, we synthesized five types of Au25SR18 NCs with different surface ligands, as shown in Figure 1a. Type I (represented in black) refers to Au25 NCs protected by an alkane-thiol (i.e., 6-mercaptohexanoic acid (MHA)), which only contains one carboxylic group, –COOH. Type II (represented in brown) refers to Au25 NCs protected by an aromatic-thiol (i.e., pmercaptobenzoic acid (MBA)), which also only contains one carboxylic group, –COOH. Type III (represented in red) refers to Au25 NCs protected by an amino acid with thiol


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group (i.e., cysteine (Cys)), which contains one carboxylic and one amine group, –COOH and –NH2. Type IV (represented in purple) refers to Au25 NCs protected by bithiolate ligands, MHA and cysteamine (Cystm) at three different ratios; where MHA contains one carboxylic group –COOH, and Cystm contains one amine group –NH2. Type V (represented in blue) refers to Au25 NCs protected by bithiolate ligands, MHA and 2-mercaptoethanol (MetH) at three different ratios; where MHA contains one carboxylic group –COOH, and MetH contains one hydroxyl group –OH. Owing to the precise control in synthesis, a library of various Au25 NCs protected by different type and composition of ligands has been produced. This family of Au NCs with controlled surface chemistry provides a good platform to understand the effect of surface properties of Au NCs on their antimicrobial activity, as comparative models on charge and functional groups effects. We first used UV-vis absorption spectroscopy to characterize the as-synthesized five types of Au25 NCs protected by different thiolate ligands. As shown in Figure 1b, a main absorption peak at ~670 nm along with two shoulder peaks at ~440 and ~780 nm were observed in all UV-vis absorption spectra. These characteristic peaks suggest that the as-synthesized Au NC samples are Au25SR18 rather than bigger sized Au NPs (typically with a characteristic surface plasmon resonance (SPR) peak at ~520 nm) or other sized Au NCs in solution.36 Some slight deformations of UV-vis absorption spectra were seen for type IV and V Au NCs, which contained a high ratio of Cystm (Figure 1b, x = 6-9, purple line) or MetH (Figure 1b, x = 7-10, blue line). This observation could be attributed to the co-localization of positively charged –NH3+ or neutral –OH with the negatively charged –COO-. Co-localization of two opposite charged groups on the Au NC surface would lead to a surface charge anisotropy, which could result in a slight distortion of their cluster structures in solution.36, 45 Despite the slight distortion in cluster structure, we have confirmed that these Au NC samples are still Au25SR18 instead of other sized Au NCs.36, 45 We further used electrospray ionization mass spectrometry (ESI-MS) to verify the molecular formula of the assynthesized Au NCs, where all five types of Au NC products were confirmed to be Au25SR18 (Figure 1c). Taking Type I Au25MHA18 as an example (Figure S2, top panel), three sets of peaks at m/z 1262, 1514, and 1892 were observed, which correspond to [Au25MHA18 - 5H]6-, [Au25MHA18 - 4H]5-, and [Au25MHA18 - 3H]4-, respectively. Therefore, only one species Au25MHA18 was obtained, suggesting the good purity of the NC products. The zoom-in spectrum of [Au25MHA18 - 4H]5- was shown in Figure 1c, black line. The peak at m/z 1514 is [Au25MHA18 - 4H]5-, and the following peaks are from the successive coordination of [+ Na - H] of [Au25MHA18 - 4H]5- (see detailed analysis in Figure S2, middle panel). Similarly, other mass spectra in Figure 1c are zoom-in spectra of [Au25SR18 - 4H]5- protected by the other four types of ligands (see detailed ESI-MS spectra in Figure S2-S10, top panels). For mono-thiolate-protected type II

and III Au25 NCs, they are confirmed to be Au25MBA18 (Figure 1c, brown line; the peak at m/z 1540 is [Au25MBA18 5H + Na]5-, see detailed analysis in Figure S3) and Au25Cys18 (Figure 1c, red line; the peak at m/z 1417 is [Au25Cys18 - 5H + Na]5-, see detailed analysis in Figure S4). For bi-thiolate-protected type IV and V Au25 NCs, the composition of surface ligand was adjusted through tuning the adding ratio of two types of ligands. In general, the ratio of MHA to Cystm (or MetH) is 19/1, 7/1, and 5/3, respectively, to form Au25CystmxMHA18-x (or Au25MetHxMHA18-x, x represents the number of Cystm or MetH). As shown in the ESI-MS data, the lower of the MHA to Cystm (or MetH) ratio, the more Cystm (or MetH) could be doped inside Au25SR18 in solution. For MHA/Cystm = 19/1, Au25CystmxMHA18-x (x = 0-2) was synthesized (Figure 1c, the first purple line), where the m/z 1486, 1500, and 1514 represent Au25Cystm2MHA16, Au25Cystm1MHA17, and Au25Cystm0MHA18, respectively (see detailed analysis in Figure S5). For MHA/Cystm = 7/1, Au25CystmxMHA18-x (x = 1-4) was synthesized (Figure 1c, the second purple line), where the m/z 1458, 1472, 1486, and 1500 represent Au25Cystm4MHA14, Au25Cystm3MHA15, Au25Cystm2MHA16, and Au25Cystm1MHA17, respectively (see detailed analysis in Figure S6). For MHA/Cystm = 5/3, Au25CystmxMHA18-x (x = 6-9) was synthesized (Figure 1c, the third purple line), where the m/z 1387, 1401, 1415, and 1429 represent Au25Cystm9MHA9, Au25Cystm8MHA10, Au25Cystm7MHA11, and Au25Cystm6MHA12, respectively (see detailed analysis in Figure S7). Similarly, for MHA/MetH-protected Au25 NCs, when the ratio of MHA to MetH was varied from 19/7, 7/1 to 5/3, the Au25MetHxMHA18-x (x = 0-2), Au25MetHxMHA18-x (x = 1-5) and Au25MetHxMHA18-x (x = 7-10) were synthesized, respectively (three blue lines in Figure 1c, see detailed analysis in Figure S8-S10). These characterization data showed that we have precisely manipulated the surface ligands on Au25SR18. While the Au core remains the same (25 Au atoms), the only difference between the above mentioned five types of Au25 NCs is the surface ligands with different functional groups. One key difference in the ligands used in this study is their charge bearing functional groups. Owing to the difference in the pKa values (Table S2) of the functional group –COOH, –NH2 and –OH, and the composition of these groups on the NC surface, Au25 NCs could carry different overall surface charges in solution. This could be clearly observed from the zeta potential measurements (Table 1) of the assynthesized Au NCs protected by different ligands.

Table 1. Zeta potential of different ligand-protected Au25 NCs in water.

Au25 NCs Au25MHA18 Au25MBA18 Au25Cys18 Au25CystmxMHA18-x


x =0-2 x = 1-4

Zeta Potential (mV) -37.4 ± 2.5 -36.8 ± 3.2 -20.8 ± 2.8 -32.2 ± 3.3 -29.5 ± 3.8


Au25MetHxMHA18-x

x = 6-9 x = 0-2 x = 1-5 x = 7-10

-20.5 ± 4.1 -38.7 ± 2.3 -36.6 ± 4.8 -33.7 ± 3.1

In order to explore the effects of surface ligands on Au NCs on their antimicrobial ability, we studied the killing effect of the five types of Au25 NC variants protected by different ligand types. One commonly used Gram-positive bacteria, Staphylococcus aureus (S. aureus), was chosen as the bacterial model. The S. aureus was treated with five types of Au25SR18 of the same concentration (and also the same concentration of Au25 NCs (molecules) in solution as Au NCs used in this study have the same Au atom number; 0.1 mM on the basis of Au atoms). This is one of the advantages of Au NCs over their larger counterparts Au NPs, that is, their atomically precise property leads to quantification treatment. To analyze the killing efficacy of each type of Au25 NCs, we utilized SYTOX green nucleic acid stain dye to differentiate live and dead bacteria. As SYTOX green is inherently cell impermeant, it could only penetrate into cells with a compromised membrane. Therefore, the positively stained cells are considered as dead cells.48 In addition, a cellpermeant nuclear stain Hoechst 33342 could be used to stain the total bacteria population (live and dead), and this data can be used to normalize the percentage of the dead bacteria. The representative integrated fluorescence images of the bacteria treated by the five types of Au25 NCs for 2 h were shown in Figure S11-S13, and the merged and enlarged fluorescence images were shown in Figure 2a-c. The percentage of dead bacteria after treatment was calculated and depicted in Figure 2d.

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In good agreement with our previous report, we observed that the Au25MHA18 NCs have effectively killed ~95% of S. aureus (Figure 2a, black column of Figure 2d).31 Moreover, Au25MBA18 that carries similar monotypic functional group –COOH, exhibited a similar killing effect as that of Au25MHA18, whereby ~93% of S. aureus population was effectively killed (Figure 2a, brown column of Figure 2d). This data further proved the effective antimicrobial ability of Au25 NCs. In contrast to Au25MHA18 and Au25MBA18 variants, Au25Cys18 dramatically lost the NCs’ effective antimicrobial properties, and only ~15% of S. aureus population was registered as dead (Figure 2a, red column of Figure 2d). This data suggests the types of surface ligands could influence the antimicrobial ability of Au25 NCs. As mentioned above, the only difference of Au25Cys18 with Au25MHA18 and Au25MBA18 is the surface ligand, viz. the surface functional groups of the Au25 NCs. As MHA and MBA only contain one functional group, –COOH, the assynthesized Au25MHA18 and Au25MBA18 are protected by 18 –COOH groups on their respective NC surface. However, Cys carries two functional groups, –COOH and –NH2, resulting in Au25Cys18 with 18 –COOH as well as 18 –NH2 on its surface. The difference in surface functionalities of Au NCs led us to further ask whether the presence of –NH2 group on NC surface would compromise the antimicrobial ability of Au25 NCs.

Figure 1. Characterization of the as-prepared Au25 NCs protected by different thiolate ligands. The molecular formula of each Au25 NC was indicated in the image. (a) Simplified structure of each Au25 NC. Color scheme: yellow, gold; grey, sulfur; black, carbon; blue, oxygen in –COOH group; red, nitrogen in –NH2 group; green, oxygen in –OH group. The structure was simplified by projecting only 6 thiolate ligands instead of the total 18 thiolate ligands. The ratio of different thiolate ligands was indicated in the image. The crystal structure of thiolate-protected Au25 NCs was adapted ACS Paragon Plus Environment from ref 47, reprinted with permission. Copyright 2008 American Chemical Society. (b) UV-vis absorption and (c) ESI-MS spectra of each Au25 NC synthesized in this study.

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Figure 2. Au25 NCs protected by thiolate ligands bearing higher ratio of –COOH groups showed better bacterial killing efficiency. (a-c) Merged and enlarged representative fluorescence images of the S. aureus after 2 h treatment of Au25 NCs. The dead cells were stained by SYTOX green (false color: red), while the total cells were stained by Hoechst 33342 (blue). Scale bar is 25 μm. (d) Percentage of the dead S. aureus after treated with different ligand-protected Au25 NCs for 2 h. Data are means ±S.D., n = 3, Student’s t-test; * significant against the water-treated group, p < 0.05; # significant against the water-treated group, p < 0.001.

To verify the effect of functional group –NH2, we used Cystm with –NH2 to form Au25CystmxMHA18-x. As shown in Figure 1, Au25 NCs bearing different ratios of Cystm and MHA on their surface were synthesized. Since Cystm only contains one functional group –NH2, and MHA only contains one –COOH, the number x of Cystm shows the corresponding number of x –NH2 and 18-x –COOH outside the Au25CystmxMHA18-x NCs. For example, Au25CystmxMHA18-x (x = 6-9) is protected by 6-9 –NH2 groups and 9-12 –COOH groups on its surface. We observed inverse relationship between the number of –NH2 groups on the Au25 NC surface and their antimicrobial efficacy. As shown in Figure 2b and purple columns of Figure 2d, the more Cystm being incorporated on the Au25 NC surface, the lower the antimicrobial ability of Au25 NCs. The percentage of dead bacteria was dropped from ~91% to 28% and ~16% with the number of –NH2 groups on the Au25 NCs increased from 0-2 to 1-4 and 6-9, and the number of –COOH group decreased from 16-18 to 14-17 and 9-12. This data is consistent with the killing behavior of Au25Cys18, indicating that more –NH2 and less –COOH could nullify bacterial killing efficiency of

Au25 NCs. We further asked whether this diminishing antimicrobial activity was solely caused by the presence of NH2 groups on the Au25 NC surface. To address this issue, we doped another surface ligand MetH (with –OH) on the surface of Au25 NCs. As the incorporation of MetH could decrease the number of –COOH on the NC surface, the resultant Au25MetHxMHA18-x carried both –COOH and –OH groups on the NC surface. As shown in Figure 2c and blue columns of Figure 2d, the influence of –OH group towards killing efficiency of Au25 NCs was not as significantly decreased as those with –NH2 group. While the number of –OH group on NC surface was increased from 0-2 to 1-5, and the number of –COOH group on NC surface was decreased from 16-18 to 15-17, the killing efficiency of Au25 NCs was only decreased from 92% to 90%. Dramatic reduction in killing efficiency, which reached ~38%, was observed when the number of –OH group was 7-10 and the number of –COOH group was 8-11, and lesser –COOH groups on the Au25 NC surface would compromise their antimicrobial ability. Consistently, the antimicrobial data suggest that –COOH group



on the NC surface would promote the Au25 NCs’ bacterial killing efficiency. The higher ratio of –COOH group on the surface of Au25 NCs, the better antimicrobial ability of Au25 NCs. As shown in our control experiment in Figure S14, the five ligands (MHA, MBA, Cys, Cystm, and MetH) did not show any bacterial killing ability. This suggests that the observed antimicrobial properties could not be due to the ligand itself, and it should be the result of Au NCs as an entire entity. Similar effects were also proved in Gram negative E. coli as shown in Figure S15. In general, nanomedicine needs to be internalized by the cells, which could further induce the cellular response. Moreover, surface ligands are known to be able to modulate NPs’ uptake by the cells.1, 5, 6 It is possible that the difference in the antimicrobial efficacy between the five types of Au25 NCs was due to their inherent difference in cell uptake. As such, we measured the internalization of the Au25 NC variants. Figure 3 showed that the bacterial cells would internalize more Au25 NCs protected by surface ligands bearing –NH2 groups, when compared to those bearing – COOH groups. Au25Cys18 and Au25CystmxMHA18-x (x = 6-9) uptake was registered to be ~1.4 and ~1.5 fold higher than the Au25MHA18 (Figure 3a and 3b). The increase in cellular uptake observed in the –NH2 bearing Au25 NCs suggests that the positively charged group on the Au25 NC surface could facilitate their initial interaction with the cells and their subsequent internalization. It is worth noting that although –NH2 group could promote the internalization of Au25 NCs inside the cells, the increased internalization of Au25 NCs did not enhance the overall killing efficacy of the Au25 NCs (Figure 2d, red and purple columns). This data suggests that the different killing efficacy observed in this study was not mainly caused by the differences in Au25 NCs internalization.

Figure 3. Uptake of Au NCs in S. aureus after treated with Au25 NCs bearing different surface ligand for 2 h. Data are means ±S.D., n = 3, Student’s t-test; * significant against the Au25MHA18 treated group, p < 0.05.

In our previous study, the antimicrobial ability of Au NCs was achieved through ROS generation, which could interfere the regular bacterial metabolism and subsequently kill the cells.31 Organic ligands on materials surface also have the capability in modulating the intracellular ROS production in the cells.29, 30 We postulated that the different surface ligands on the Au NCs could induce different ROS production, leading to different antimicrobial killing effect. To better understand the role of surface ligands in determining the antimicrobial efficacy of Au25 NCs, we exposed the cells to our library of Au25 NC variants and measured their intracellular ROS production. As shown in Figure 4a,

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Au25MHA18 induced about 2.5-fold increase of the intracellular ROS production when compared to the control group (black column), which is consistent with our previous observation.31 Similarly, Au25MBA18 induced ~2-fold increase on the ROS production (brown column). These increased ROS level corresponds well with the high antimicrobial ability of the two variants of Au25 NCs. The Au25Cys18 treated group, on the other hand, barely showed any ROS level change compared to the control group (red column). In addition, as shown in Figure 4b and 4c, the trend of ROS generated by Au25CystmxMHA18-x and Au25MetHxMHA18-x NCs also fits well with their bacterial killing efficiency, whereby the higher ratio of –COOH group on Au25 NC surface, the more ROS being generated. Similar ROS results were also shown in E. coli (Figure S16). The influence of different ligand presence on Au NCs towards ROS generation ability seems to be the key determinant in their antimicrobial ability. Consistent with previous antimicrobial control experiments shown in Figure S14, the ligand alone treatment resulted in minimal increase (~10%) in the ROS production profile (Figure S17). This data further proves that the entire entity of the Au NCs was required to generate ROS.

Figure 4. Au25 NCs protected by thiolate ligands bearing higher ratio of – COOH groups would induce higher intracellular ROS production. Relative intracellular ROS production level of S. aureus following treatment of different ligand-protected Au25 NCs for 2 h. The intracellular ROS level for the water-treated group was set as 1. Data are means ±S.D., n = 3, Student’s t-test, * significant against the water-treated group, p < 0.05; # significant against the water-treated group, p < 0.001.

As ROS is a byproduct of intracellular redox metabolism in the cell, it is possible that the difference in the ROS production is the result of the different redox response towards any Au25 NCs introduced into the system.49, 50 As such, we investigated the effect of the highest ROS inducers (i.e., Au25MHA18 and Au25MBA18) and the lowest ROS inducer (i.e., Au25Cys18) on the intracellular oxidative process. Genes (i.e., dmpI, narJ and narK), which are responsible for the expression of metabolic enzymes to promote the oxidation process, were utilized to measure the Au25 NCs’ induction effect. dmpI gene encodes 4-oxalocrotonate tautomerase, which is responsible for the oxidative catabolism of toluene, o-xylene, 3-ethyltoluene, and 1,2,4trimethylbenzene into their intermediates of the citric acid cycle, producing ROS as its enzymatic by-product.51 narJ gene is related to the respiratory nitrate reductase, which could transfer electrons from NADH or NADPH to nitrate.52 narK gene is related to the nitrate transport coupled to nitrate reduction by nitrate reductase.52, 53 As shown in Figure 5, these pro-oxidative genes were all up-regulated


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following the Au25 NCs treatment. In good agreement with our ROS production data (Figure 4a), the cells treated with Au25MHA18 and Au25MBA18 showed high gene expression, registering 2.0-fold and 1.4-fold expression of narJ (Figure 5b). The Au25Cys18, however, induced up-regulation of these genes in the cells in much lesser degree, and the comparative Au25Cys18 induced narJ expression registered at 1.1-fold (Figure 5b). This suggests that the surface ligands on Au25 NCs could induce the intracellular ROS production through modulation of pro-oxidative enzyme. However, we cannot exclude the possibility that these AuNCs might drive redox reactions in bacteria as oxidase and peroxidases like “pseudo-metalloenzymes”. It is worth noting that the Au25Cys18 actually induced the up-regulation genes that encode the pro-oxidative enzyme,

positive charges.55, 56 AMP utilizes the positive charge groups to initiate the interaction with the cells that allows it to insert its hydrophobic tail to the bacterial cell wall, resulting in the bacterial cell damage and a subsequent cell death.57, 58 Nevertheless, the key working mechanism for Au NCs is generating ROS to kill bacteria.31 Our results thus far indicate that the positively charged groups that we have incorporated on the surface of Au25 NCs could facilitate the initial interaction with the bacterial cells, but they could be detrimental to the ROS inducing capability of the Au25 NCs. Scavenging capability shown by the positively charged functional groups suggest that these functional groups might not be the best option to be incorporated on the metal NPs-based antimicrobial agents that rely on the ROS production to exert their antimicrobial activity. In effort to balance the internalization necessity and the redox preserving capability, nanotechnologists should carefully consider the surface ligand variants and composition in their design of metal NPs-based antimicrobial agents.

Figure 5. Au25 NCs promote cellular oxidation process in S. aureus. Increased expression of the oxidation regulating genes: (a) dmpI, (b) narJ and (c) narK, was observed after treatment with Au25MHA18, Au25MBA18 and Au25Cys18 for 2 h. Gene expression of the water-treated group was set as 1. Data are means ±S.D., n = 3, Student’s t-test, * significant difference against the water-treated group, p < 0.05.

albeit in much lesser degree compared to Au25MHA18 and Au25MBA18. Considering this, one could argue that with these genes up-regulation, Au25Cys18 should be able to produce measurable ROS level. Nevertheless, Au25Cys18 treated cells showed even lower ROS production level (Figure 4a) when compared to the control group, which is ~0.93-fold. This depletion on the ROS production level following Au25Cys18 treatment was possibly caused by the redox buffering capacity of the functional groups. As positively charged –NH2 could quench singlet oxygen, their presence on the surface would deplete the produced ROS.54 As such, the more moieties of positively charged –NH2 were introduced on the surface, the further the ROS production were depleted, reducing the overall ROS bioavailability to exert antimicrobial effect (Figure 2b and 4b). In contrast to the positively charged groups, the negative –COOH could better preserve the generated ROS, resulting in Au25 NCs protected with these groups to have the best antimicrobial performance among the rest of other surface groups (Figure 2a and 4a). Therefore, the antimicrobial ability would also decrease when –COOH was buffered by neutrally charged – OH, as their ROS preserving capability was weakened (Figure 2c and 4c). Moreover, our results in Au NCs showed deviations from the well-known paradigm, in which more positively charged antimicrobial agents show higher bacterial killing efficiency.23, 27 We reckoned this contrast was caused by the different killing mechanisms between the Au NCs and the amphipathic AMPs that were commonly known to bearing

Figure 6. Au25 NCs could penetrate through the biofilm, and Au25MHA18 showed better killing performance compared to the Au25Cys18 in the bacterial spheroids model. (a) Schematic illustration of 3D bacterial spheroids image acquisition and reconstruction. (b) Representative fluorescence images of the bacteria spheroids after 2 h treatment of water, Au25MHA18 and Au25Cys18. The dead cells were stained by SYTOX green (false color: red), while the total cells were stained by Hoechst 33342 (blue). Scale bar is 100 μm.



In in vivo settings, bacteria cells form their community within the protection of biofilm barrier.59-61 Antimicrobial agents, therefore, need to penetrate into this biofilm barrier to kill the bacterial cells that reside inside the barrier. In order to prove the efficacy of the Au NCs in a more realistic setting, we constructed bacterial bead spheroids in which agar was used to embed the bacteria cells as well as mimic the polymer network within the biofilm. We then tested out the efficacy of the most negatively charged Au25MHA18, as well as the least negatively charged Au25Cys18 on the spheroids model (the imaging acquisition and reconstruction was shown in Figure 6a) As shown in Figure 6b and Figure S18-S20, Au25MHA18 could effectively kill the bacteria within the spheroids, while Au25Cys18 barely showed antimicrobial ability. This data is in good agreement with the killing effect we observed when those individual cells are in suspension (Figure 2a). As Au NCs killing efficacy hinges on their internalization to the cells, the cell death signals from the Z-stack layers could be used to determine the penetration degree of the Au NCs (Figure S19 and S20). Our Z-stack image acquisition showed that Au25MHA18 and Au25Cys18 could all penetrate through the spheroids and reach their center. This penetration was evidenced from the SYTOX green signal that was observed at the center of the spheroids, which was captured approximately in Zstack image layer 7-8. Considering that each Z-plane was taken at 5 μm interval, we could estimate that the Au NCs could penetrate ~30 μm biofilm thickness. This data suggests that Au NCs could penetrate through the biofilm barrier and exert their antimicrobial efficacy even on the biofilm-protected bacterial cells, which offers an advantage over large Au NPs. Moreover, this model in spheroid culture brings Au NCs based antimicrobial agents one step closer to real world applications.

CONCLUSION In conclusion, we synthesized five types of Au25SR18 NCs protected by different type and ratio of surface ligands. It was found that the charge of the functional groups could determine the surface charge of Au25 NCs, and Au25 NCs contained more negative –COOH groups hold more negative zeta potential. The surface charge of Au25 NCs could influence their ROS generation ability, and more negatively charged surface would better preserve the as-generated ROS, leading to higher overall ROS level. Therefore, more ROS would interfere the metabolism enzyme of bacteria, disturb their normal metabolism, and kill the bacteria in the end. Our finding is different from the common belief that more positively charged antimicrobial agents would show better antimicrobial behavior. This finding indicates the different role of surface ligands on nanomaterials in exerting the antimicrobial properties. Our work suggests an alternative design of antimicrobial agents based on this different working mechanism.

ASSOCIATED CONTENT Supporting Information. Experimental section of bacterial related tests like cell death analysis, ROS measurement, up-

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take measurement, relative gene expression, and bacterial spheroids death analysis could be found in supplementary information. In addition, supplementary data which include TEM images, DLS test, ESI mass spectra, representative fluorescence images, Au NCs uptake data, representative Z-stack images of the bacterial spheroids, etc. are also given in Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (J. Xie); [email protected] (D. T. Leong); [email protected] (M.I. Setyawati)

ACKNOWLEDGMENT This work was financially supported by Ministry of Education, Singapore, under the grant R-279-000-481-112. K. Zheng acknowledges the National University of Singapore for her research scholarship.

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