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Antimicrobial Cluster Bombs: Silver Nanoclusters Packed with Daptomycin Kaiyuan Zheng,† Magdiel I. Setyawati,*,† Tze-Peng Lim,‡,§ David Tai Leong,*,† and Jianping Xie*,† †

Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore ‡ Department of Pharmacy, Singapore General Hospital, Outram Road, Singapore 169608, Singapore § Office of Clinical Sciences, Duke−NUS Medical School, 8 College Road, Singapore 169857, Singapore S Supporting Information *

ABSTRACT: Integration of two distinctive bactericides into one entity is a promising platform to improve the efficiency of antimicrobial agents. We report an efficient antimicrobial hybrid formed through conjugating silver nanoclusters (AgNCs) with daptomycin. The as-designed antimicrobial hybrid (D−AgNCs) inherits intrinsic properties of both bactericides with an enhanced synergistic performance. In particular, the chemically integrated D−AgNCs showed improved bacterial killing efficiency over the physically mixed daptomycin and AgNCs (D+AgNCs). More interestingly, the as-designed D−AgNCs could effectively damage the bacterial membrane. Propidium iodide (PI) stain showed bacterial membrane damage in about 85% of the bacteria population after treatment with D−AgNCs through creation of larger pores on the membrane as compared to D+AgNCs, largely due to the localization of daptomycin within the hybrid structure. These larger pores facilitated the entry of the D−AgNCs into the cell and led to more severe DNA damage of the bacterial DNA as compared to D+AgNCs in genomic DNA PAGE analysis. TUNEL assay further depicted more bacterial DNA breaks induced by D− AgNCs. The RecA gene expression level was upregulated, suggestive of DNA repair activation. The strong induced DNA damage benefited from the localization of AgNCs in the core of the antimicrobial hybrid structure, which could generate localized high ROS concentration and work as a critical ROS reservoir to continually generate ROS within the bacterium. The continual bombardments by these ROS generators restrict the ability of the bacteria to now develop resistance against this. KEYWORDS: silver nanoclusters, metal nanoclusters, daptomycin, antibiotics, antimicrobial hybrid resistant Enterococci).10 Resistance against this drug was recorded as low as 0.2% for all clinical trial cases.10 Nevertheless, this number would not remain stagnant. In addition, current clinical practice requires an extended therapy time of 20 days. Therefore, due to unfavorable pharmacokinetics, this drug could only reach the infection site at a suboptimal dose.11 Daptomycin exerts its bactericidal effect

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ntimicrobial agents are constantly playing catch-up with multidrug resistance (MDR) bacteria.1−3 At the current slow pace of antimicrobial agent development, the world may one day be facing an epidemic of untreatable bacterial infections of cataclysmic proportions.3−7 This fear motivates the development of efficient antimicrobial agents and even efficient paradigms of delivery and circumventing antibiotics resistance.8,9 Currently, daptomycin is one of the remaining few antibiotics with a certain clinical efficacy in combating the multidrug resistance Gram-positive bacteria (e.g., methycylin-resistant Staphylococcus aureus and vancomycin© 2016 American Chemical Society

Received: June 10, 2016 Accepted: August 5, 2016 Published: August 5, 2016 7934

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Figure 1. (a) UV−vis absorption (solid lines) and photoemission spectra (dashed lines, λex = 489 nm) of the as-prepared AgNCs (black lines) and D−AgNCs (red lines) in water. The inset shows photographs of the as-prepared AgNCs and D−AgNCs in water under visible (two items in the left panel) and UV light (two items in the right panel) in sequence. (b) Hydrodynamic size distribution of the as-prepared AgNCs (black) and D−AgNCs (red).

molecules, giving these NCs a better interface and interaction with the subcellular components (e.g., cell membrane, DNA, and protein), which could perturb their functions more effectively with the designed end target of bactericidal effect.32,33 Being in an AgNC format also allows a slower but more sustained release of Ag species, distinctively different from a burst release behavior when Ag+ ions were directly used. The sustained release could also protect the mammalian cells from unnecessary spikes of toxicity due to the Ag+ ion pulse, therefore minimizing their potential cytotoxicity in the final in vivo setup.34 We therefore hypothesized that by packaging AgNCs (an emerging class of inorganic antimicrobial agents) together with daptomycin (a commercial organic antibiotic) we could achieve a synergistic package (hybrid) against bacteria. Here, we designed an efficient hybrid of bactericides comprising AgNCs and daptomycin, conjugated together through a strong covalent bond (amide). Because of their ultrasmall size, the AgNCs could be considered as small drug molecules within the same size range of daptomycin, allowing both of them to be mixed and conjugated homogeneously in a well-controlled manner in the hybrid. This homogeneous conjugation ensures a more consistent release of both agents, which is a key requirement if a synergistic effect is required from the hybrid. The lack of a consistent release of both agents would negate any synergistic effects since each agent will be acting differently and temporally. The integration of AgNCs with daptomycin into one hybrid allows the NCs to have their high surface area for intracellular ROS generation and favors the daptomycin to be highly localized on the NC surface for an optimum membrane disruption via its lipophilic tail insertion. In comparison, if the bulkier Ag nanoparticles were used in conjunction with the daptomycin, one could expect the formation of a daptomycin layer outside the Ag nanoparticles, which could reduce the overall antimicrobial efficiency of the hybrid. Therefore, through our hybrid strategy, which allows an efficient and homogeneous conjugation of AgNCs and daptomycin, we could obtain a certain synergistic effect of both antimicrobial agents, thus reducing the amount of daptomycin required to treat any given infection. This effect may also largely minimize the bacteria exposure to daptomycin and subsequently on the long run curtail their likelihood to develop resistance toward the daptomycin, in general, and the hybrid structure, in particular. Here, we show that the AgNCs−daptomycin hybrid

through one known mechanism, where its lipophilic tail could cause the bacterial membrane damage.10,12 A suboptimal dose over an extended therapy time coupled with a single bactericidal mechanism have been recognized to be the common causes for bacteria to develop resistance to antibiotics.13 Thus, it is foreseeable that daptomycin will follow the eventual course of many other older antibiotics, and be rendered ineffective due to resistance. Thus, it is imperative to develop efficient strategies to prolong the otherwise short lifespan of daptomycin against the multidrug-resistant Grampositive bacteria. Combinatorial therapy of a bactericides cocktail is known to synergistically improve the performance of each agent by attacking the bacteria from two distinct fronts.14−16 This efficient approach could be presented to bacteria, a more difficult evolutionary hurdle to overcome. Silver (Ag) has been known to exert a wide-spectrum antimicrobial property through various killing mechanisms (e.g., membrane damage, DNA damage, and perturbation of cell metabolism triggered by a high reactive oxygen species (ROS) production).2,17−20 Such properties make Ag material a promising candidate for the intended combinatorial therapy with daptomycin. It should be noted that the bactericidal performance of Ag material is mainly controlled by its size.17 In particular, as the size of Ag nanoparticles decreases, their antibacterial efficacy increases due to the increased potential to release Ag atoms and ions.17,21−23 However, going to the extreme of introducing Ag+ ions together with daptomycin presents another set of problems: being able to physically combine both Ag+ ions and daptomycin as a single package. In order to fully harness the synergism and the ease in ensuring that both Ag species and daptomycin are delivered as a single package, we synthesized ultrasmall AgNCs comprising of an exact number of Ag atoms within the core and protecting ligands of glutathione (GSH) in the outer layer.24 Generally formulated as AgxLy (where L denotes the thiolate ligand), the molecular-like AgNCs possess a core size smaller than 2 nm, making them have a significantly higher surface to volume ratio compared to their bulk counterpart and Ag nanoparticles.25−30 This means that compared to the same concentration of bulk Ag or Ag nanoparticles AgNCs have more surface Ag atoms available to induce the ROS generation in addition to having more Ag atoms to be released out from the structure to kill the bacteria.24,31 The ultrasmall size feature of AgNCs also makes them comparable in size to small 7935

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Figure 2. (a) Representative fluorescence images of the bacterial cells after 2 h treatment. The dead cells were visualized by PI staining (red), while the Hoechst 33342 (blue) helped to identify all cells. Scale bar is 25 μm. (b) Percentage of PI-stained bacteria to show damaged bacterial membrane. Data are means ± S.D., n = 3, Student’s t test. Compared to the water-treated group, * is significant against the watertreated group, p < 0.05; # is significant against the water-treated group, p < 0.001. Comparison of the corresponding NAC- and non-NACtreated group; α is significant difference against non-NAC-treated group, p < 0.05. (c) Histogram of the PI intensity in each individual bacterial cell treated with D+AgNCs (black line) and D−AgNCs (red line). (d) Relative lipid peroxidation level after incubation for 2 h, where the lipid peroxidation of the water-treated group was set as 1. Data are means ± S.D., n = 3, Student’s t test. # is significant against the watertreated group, p < 0.001.

RESULTS AND DISCUSSION

(D−AgNCs) demonstrated the highest killing effect against the Gram-positive model bacteria S. aureus when compared to other control groups. In addition to demonstrating the efficacy of the as-designed antimicrobial hybrid, we also elucidated the mechanism that drives the antimicrobial hybrid’s enhanced killing effect.

In this study, we used a well-defined AgNC construct of Ag16SG9, in which each AgNC contains 16 Ag atoms and 9 glutathione (GSH) protecting ligands.31 The as-prepared AgNC was dark brown in solution with strong red emission under UV illumination. We observed the absorption peak of the as-prepared AgNCs at 490 nm. This is in line with our 7936

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AgNCs, giving a molar ratio close to 1:1 of daptomycin and AgNCs within the D−AgNCs hybrid structure. To determine the efficacy of our bactericide hybrid, we treated our model bacteria S. aureus with 200 μM of D− AgNCs. The concentration of the D−AgNCs here was determined on the basis of Ag atoms within the structure through inductively coupled plasma optical emission spectrometry (ICP-OES). Since AgNCs were in the form of Ag16(SG)9 and the molar ratio of daptomycin and AgNCs was determined to be 1:1 through TGA analysis (Figure S5), we could determine the concentration of daptomycin inside D−AgNCs to be approximately 10 μM. As such, 10 μM daptomycin and 200 μM AgNCs (on the basis of Ag atoms) were introduced to the bacteria as control groups. D−AgNCs showed enhanced antimicrobial ability, and they could effectively inhibit the growth of bacteria after treatment of 1 and 2 h (Figure S6). Daptomycin alone showed insignificant inhibitory effect on the bacterial growth, as evidenced by the similar bacterial growth fold observed both in the water-treated and daptomycin-treated groups (Figure S6). As expected, the AgNCs-treated bacteria showed a significant growth reduction of ∼80% of the bacterial growth in the water-treated group (Figure S6). Interestingly, the D−AgNCs-treated group showed an enhanced bacteria growth reduction compared to the control groups by using daptomycin or AgNCs alone, where only ∼70% of bacterial growth was obtained after 2 h of treatment (Figure S6). In comparison, bacteria treated with the same concentration of daptomycin and AgNCs mixture but without the benefit of conjugation (D+AgNCs for short) showed only ∼75% bacterial growth (Figure S6). This supports our hypothesis that the hybrid containing localized AgNCs and daptomycin could produce a synergistic effect that results in the bacteria growth reduction. It was found that the D−AgNCs hybrid could effectively damage the bacterial membrane, leading to their improvement on killing efficiency. We determined the membrane damage through a DNA-binding fluorescent dye, propidium iodide (PI). Cells with an intact membrane are not PI-permeable. As such, PI binding with DNA suggests the presence of damaged membrane. In addition, we also employed a cell-permeant nuclear stain Hoechst 33342 to stain all bacteria population regardless of being alive or dead (Figure 2a and Figure S7) as a normalizing factor. These two stains allowed us to determine the membrane-damaging degree of each treatment group (Figure 2b). It was found that the water-treated group barely showed any bacterial membrane damage, while the daptomycin- and AgNCs-treated groups showed ∼12% and 28% bacterial membrane damage, respectively. In contrast, the D +AgNCs group led to ∼54% bacterial membrane damage. D− AgNCs caused membrane damage close to 84% of bacteria population, and this is the most damaging among all the treatment groups. The D−AgNCs’ enhanced bacteria membrane damage clearly supports our hypothesis that the hybrid design could provide the synergistic effect for highly efficient bacteria killing performance and the need to homogeneously conjugate both active species together into one entity. The enhanced membrane damage could be attributed to the increased interaction that could occur between the D− AgNCs and the bacterial membrane. This was evident from the overall surface charge shift upon the daptomycin conjugation. We determined the ζ potential of AgNCs to be about −30.5 mV, while the D−AgNCs’ ζ potential was registered at a less negative value of about −22.8 mV. The ζ

previously reported AgNCs with a good antimicrobial property.31 It is worth noting that upon the conjugation of daptomycin with the AgNCs (hereafter referred to as D− AgNCs), the AgNCs inside D−AgNCs hybrid maintained their ultrasmall size instead of aggregating into larger sized nanoparticles. This is evidenced from Figure 1a in which D− AgNCs showed a red emission comparable to that of the unconjugated AgNCs in solution, although a minor red shift of its photoemission wavelength was observed. A slight shift was also detected on the absorption peak of D−AgNCs at 480 nm, which is distinctively different from the characteristic surface plasmon resonance (SPR) peaks of the large Ag nanoparticles, typically at ∼400 nm.35,36 This result further supports that the AgNCs within the hybrid structure maintained their ultrasmall structure. The minor photoemission and absorption shift shown in D−AgNCs was expected and could be contributed to the conjugation of daptomycin. Transmission electron microscopy (TEM) analysis provides more supportive evidence on the ultrasmall size of AgNCs within the D−AgNCs network (Figure S1), where ultrasmall AgNCs were clearly seen in both unconjugated (Figure S1a) and daptomycin-conjugated (Figure S1b) samples. Our metaanalysis of the daptomycin structure (Figure S2) suggests that both daptomycin and AgNCs within the D−AgNCs network are in a comparable size (∼2 nm).37 Thus, it could be logically surmised that both AgNCs and daptomycin could mix homogeneously within this hybrid network. This was also evidenced from the TEM images in which AgNCs were shown to be localized homogeneously within a ∼100 nm D−AgNCs network (Figure S1b). The daptomycin conjugation was also evident by the dramatic hydrodynamic size increase following its conjugation with the AgNCs. As shown in Figure 1b, the average hydrodynamic size of D−AgNCs hybrid was ∼200 nm, while the native ultrasmall AgNCs was below 5 nm in size. In addition to the size increase, the daptomycin conjugation also resulted in the broadening of the size distribution. The nonspecific conjugation feature of the EDC/NHS coupling method is the key factor for this polydispersity. Since both the daptomycin and glutathione on the AgNC outer layer possess multiple carboxylic and amino groups, the conjugation could occur in a random fashion, which makes controlling the final size of the network challenging, and a relatively high dispersity network structure of cross-linked daptomycin and AgNCs could be expected. To give irrefutable evidence of successful conjugation of daptomycin and AgNCs, we performed Fourier transform infrared spectroscopy (FTIR) analysis (Figure S3). We detected a prominent peak at 1520 cm−1 for the D−AgNCs. The spectrum is characteristic of the C−C stretch in the aromatic ring, and it could be attributed to the presence of benzene ring within daptomycin. A similar C−C stretch peak was observed for the daptomycin sample, but this peak was absent in the FTIR spectrum of AgNCs (protected by glutathione without a benzene group). The existence of daptomycin inside D−AgNCs was further confirmed by thermogravimetric analysis coupled FTIR (TGA-FTIR), which showed a significant decrease of the characteristic C− C stretch of daptomycin during the heating process (Figure S4). TGA analysis also allows us to measure the daptomycin content within the D−AgNCs (Figure S5). Our analysis showed that daptomycin accounted for ∼20% (w/w) in D− 7937

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Figure 3. (a) PAGE result of the extracted genomic DNA stained with SYTOX Green. (b) Representative fluorescence images of the bacterial cells after 2 h treatment. The cells were visualized by staining the DNA with Hoechst 33342 (red), while the DNA damage status of the bacterial cells was ascertained with TUNEL assay (green). Scale bar is 25 μm. (c) DNA damage index is the ratio of DNA-damaged bacteria to normal bacteria, showing the DNA damage degree. Data are means ± S.D., n = 3, Student’s t test. * is significant against the water-treated group, p < 0.05; # is significant against the water-treated group, p < 0.001. (d) Relative recA gene expression level to indicate DNA repair extent, where the values of the water-treated group and water with NAC-treated groups were set as 1. The UV-treated group was obtained by irradiating bacteria under UV light for 30 min. Data are means ± S.D., n = 3, Student’s t test. Compared to the water-treated group, * is significant difference against the water-treated group, p < 0.05. Comparison of the corresponding NAC- and non-NAC-treated group, α is significant difference against non-NAC-treated group, p < 0.05.

potential shift could be attributed to the daptomycin conjugation which by itself was measured to be about −3 mV. This ζ potential shift indicates that the interaction between D−AgNCs and the negatively charged cell membrane is more favorable when compared to the unconjugated AgNCs. This coincides well with our observation of the bacterial membrane in which the D−AgNCs showed much higher membrane damage compared to other control groups that utilized unconjugated AgNCs. The overall ζ potential of daptomycin suggests that it would allow the drug to most favorably interact with the membrane. Nevertheless, the membrane damage data suggest that the daptomycin-only treatment gave the least membrane damage compared to the other treatment groups (i.e., AgNCs, D+AgNCs, and D−AgNCs). This further highlights that the drug interaction with the bacterial membrane alone was not sufficiently effective in damaging the bacterial membrane. We hypothesized that the daptomycin−AgNCs conjugation could not only facilitate more interaction between the D− AgNCs and the bacterial membrane but also yield a localized

and therefore more concentrated daptomycin within the hybrid structure. This in turn would allow more daptomycin to be placed in close proximity to the bacterial membrane, interact with and efficiently create holes on the bacterial membrane. Moreover, the localized AgNCs inside the D−AgNCs network could generate ROS to oxidize the lipid bilayer of bacteria. To verify the involvement of ROS in membrane damage, we treated the bacteria with an antioxidant, N-acetyl-L-cysteine (NAC), for each treatment group. We hypothesized that if ROS was indeed involved in damaging the bacterial wall, NAC could reduce the efficiency of AgNCs by mitigating the oxidative stress (i.e., ROS) generated by AgNCs. It was found that NAC could effectively inhibit the membrane damage (Figure 2a and Figure S8). As shown in Figure 2b, NAC has significantly ameliorated the membrane damage caused by the D−AgNCs. This data provides a supportive evidence on the involvement of ROS in the process of membrane damage induced by the D− AgNCs. As we have identified the role of ROS in the membrane damage induced by the D−AgNCs, we further studied the 7938

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ACS Nano contribution of daptomycin in our hybrid formulation. We first decoupled the daptomycin’s contribution by isolating the ROS effect from the overall effect of the hybrid in damaging the membrane through the lipid peroxidation assay. Lipid peroxidation refers to the oxidative degradation of lipids, in which the final products are reactive aldehydes (e.g., malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE)). The reactive aldehydes would further react with thiobarbituric acid (TBA) and generate fluorescent MDA−TBA adducts. It should be noted that in our hybrid formulation ROS is produced by AgNCs instead of daptomycin. Therefore, lipid peroxidation could be used to identify the contribution of AgNCs in generating ROS. This would then allow us to decouple the contribution of daptomycin and AgNCs, and further understand the mechanistic action of our hybrid. As shown in Figure 2d, D+AgNCs showed a higher degree of lipid peroxidation than the D−AgNCs, suggesting that more ROS was generated by D+AgNCs. This is expected as the AgNCs within the D+AgNCs treatment group still retain their original cluster size, and they are able to produce higher level of ROS due to their larger surface area than the AgNCs in the D− AgNCs formulation. However, the overall membrane damage in the D−AgNCs group was significantly higher than that in the D+AgNCs group (Figure 2b), which suggests that ROS might not be the only contributing factor in the D−AgNCs. The results also indicate that the localized daptomycin moieties within the D−AgNCs group could facilitate the generation of larger and more holes on the bacterial membrane. We validated this D−AgNCs effect on the formation of bacterial membrane holes by making a comparison of the PI intensity of D+AgNCs and D−AgNCs treated bacterial cell population. It was found that the bacterial cells treated by D−AgNCs showed a right shift of PI intensity compared with D+AgNCs (Figure 2c), indicating more PI were present inside the bacteria treated by D−AgNCs. In comparison, other treatment groups showed much lower PI intensity than the D+AgNCs treated group (Figure S9). Moreover, inductively coupled plasma optical emission spectrometry (ICP-OES) analysis suggests that an increased amount of Ag was internalized by the bacterial cells after the treatment with D−AgNCs as compared to those in the control groups of D+AgNCs and AgNCs (Figure S10), which also implies that more D−AgNCs were present in the cells. This data further suggests that D−AgNCs have facilitated the generation of larger and possibly more holes on the bacterial membrane, which might make the diffusion of D−AgNCs into the cells more easily. The internalized D−AgNCs could further induce damage to the bacterial subcellular components, subsequently leading to bacterial cell death. Taken together, the above experimental evidence further highlight the advantages of the integration of daptomycin with AgNCs into one formulation. Following the extensive membrane damage, the D−AgNCs hybrid could go inside the bacteria easily, interact with the bacterial DNA, and induce the DNA damage. To investigate this, we extracted out the bacterial genomic DNA after treatment, stained it with intercalating fluorescent dye SYTOX Green, and conducted the polyacrylamide gel electrophoresis (PAGE) analysis, as shown in Figure 3a. Only the water-treated bacteria showed a compact DNA band, indicating there was no DNA damage derived from this treatment group. In contrast, all other treatment groups showed a certain degree of smearing on their DNA bands, indicating the presence of shorter genomic DNA species and DNA

damage caused by the treatment. Though all treatment showed a certain degree of DNA damage, the D−AgNCs-treated group induced the highest DNA damage, as evidenced by the longest smear detected in the PAGE analysis. In addition, DNA fragmentation was quantified with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.38 It was found that D−AgNCs hybrids caused serious bacterial DNA fragmentation. TUNEL assay works by incorporating the modified dUTPs at the 3′-OH ends of fragmented DNA through the enzyme terminal deoxynucleotidyl transferase (TdT), which would then tag bacteria with damaged DNA. The bacteria with damaged DNA were stained green after the TUNEL assay treatment, while the bacteria without DNA fragmentation were stained with Hoechst 33342 and artificially reassigned to red for better contrast (Figure 3b). The TUNEL-stained bacterial cells and Hoechst 33342 stained bacterial cells were counted individually. We determined the ratio of DNA-damaged bacteria (named as “DNA damage index”) by calculating the ratio between the TUNEL stained bacterial cells and Hoechst 33342 stained bacterial cells across three images of each group (Figure 3c). The DNase I treated group was used as control, and the DNA damage index was ∼0.53. Water and daptomycin treated groups barely showed any DNA damage, and the DNA damage index was therefore 0. This data is expected as the main action mode of daptomycin is to disturb the bacterial membrane but not eliciting damage on genomic DNA. As for the AgNCs-treated group, the DNA damage index was ∼0.09, suggesting that the AgNCs do exert a minimal damage to the cell membrane and also damage the bacterial DNA. In comparison, in the D+AgNCs group, a robust DNA damage can only occur from the first damaging the cell membrane and allows a flood of AgNCs to enter the cell, resulting in a DNA damage index of ∼0.26. It becomes even more damaging when both AgNCs and daptomycin were delivered as a single package with a DNA damage index reached ∼0.4 (Figure 3c). Moreover, the intracellular DNA damage could also be detected through the activation of DNA repair gene, recA in the bacterial cells. A quantitative reverse transcription polymerase chain reaction (RT-PCR) showed that D−AgNCs induced the highest recA gene expression level (Figure 3d), which was 2fold increase compared with the water-treated group, suggesting an intense repair of DNA was initiated. We further examined the role of ROS in this important DNA damage process. We utilized the NAC in conjunction of our treatment and detected the expression level of recA. If ROS was the key factor for the DNA damage, the removal of the generated ROS by NAC should reduce the DNA damage level in the bacteria. It was found that the NAC treatment has significantly reduced the bacterial DNA damage level in the D−AgNCs treated group. This was evidenced by the recA expression level, which was close to the basal level (Figure 3d), suggesting that the ROS generated by the internalized AgNCs was the key source for the observed DNA damage. Taking all these data together, we conclude that D−AgNCs could induce the highest DNA damage in the bacteria cells. The increase in DNA damage also supports our initial observation of the cell membrane damage elicited by the D−AgNCs. It could be argued logically that the more extensive the damage on the bacterial cells, the more hybrid structure to enter the cells and to cause more damage on the genomic DNA in a positive self-reinforcing chain of events. We have identified that ROS is involved in injuring the subcellular components like DNA (Figure 3) and cell 7939

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antimicrobial hybrids to enter the cell, producing more intracellular ROS and subsequently inducing more extensive DNA damage. Following this rationale, we should expect that there were less amount of AgNCs from the combinatorial D +AgNCs group entering the cells, and subsequently, the rate of intracellular ROS of D+AgNCs group should be lower than the D−AgNCs-treated group. Nevertheless, we observed a similar intracellular ROS production level between the two groups. This could be caused by the difference in the surface area available for the ROS generation between the two treatment groups. The AgNCs in the combinatorial D+AgNCs group were still in their individual cluster state, allowing them to have a larger surface area to induce the intracellular ROS production compared to the D−AgNCs. As such, their large surface area could overset the fact that there was lesser amount of AgNCs entering the cells in the D+AgNCs when compared to the D− AgNCs, resulting in a similar net amount of intracellular ROS observed in these two groups. More importantly, both treatment groups (i.e., D+AgNCs and D−AgNCs) induced a similar level of intracellular ROS production, yet they triggered a different level of DNA damage (Figure 3). If we assumed that ROS was responsible for the DNA damage, then a similar level of ROS within the bacteria cell should produce the same DNA damage profile between the two groups, suggesting that the ROS produced by the D−AgNCs group was more efficient in damaging the DNA. The increase in the ROS efficiency could be linked to the fact that AgNCs were localized within the D− AgNCs hybrid network. This arrangement would allow them to locally catalyze the ROS generation and release them in a high localized concentration, locally damaging the DNA and triggering the bacterial death due to the extensive localized DNA damage. In contrast, the unbound AgNCs within the D +AgNCs group did not have the benefit of localizing their ROS production site. This means though the produced ROS could damage the DNA, the damage would potentially be spread out along the bacterial DNA and never reach the cell-death inducing threshold.

membrane (Figure 2). One possible ROS generation source is the AgNC itself, in which the AgNC is able to catalyze the ROS production independent of the presence of the bacterial cells. To validate this notion, we conducted an abiotic ROS measurement (Figure S11), and we did not detect any abiotic ROS formation on all treatment groups, invalidating our initial notion that the ROS originated from the AgNC-independent catalytic activity. This also suggests that AgNC requires the presence of the bacteria to facilitate the catalytic process that could produce ROS. Thus, we further analyzed the intracellular ROS production to elucidate its role in the D−AgNCs’ enhanced bactericidal performance (Figure 4). We did not

Figure 4. Relative ROS level by normalizing the ROS concentration to cell number after incubation for 2 h, where the ROS values of the water-treated group and water with NAC-treated group were set as 1. Data are means ± S.D., n = 3, Student’s t test. Compared to the water-treated group, * is significant against the water-treated group, p < 0.05. Comparison of the corresponding NAC- and non-NACtreated groups, α is significant difference against non-NAC-treated group, p < 0.05; β is significant against non-NAC-treated group, p < 0.001.

CONCLUSION In conclusion, we developed an efficient antimicrobial hybrid through AgNCs and daptomycin conjugation. The as-designed antimicrobial hybrid showed a highly efficient killing effect. We attributed this efficient killing effect to the fact that the D− AgNCs hybrid could localize AgNCs as well as daptomycin within the network. Localized daptomycin inside the hybrid structure could effectively damage the bacterial membrane, while the localized AgNCs could generate ROS to oxidize the bacteria lipid bilayer to further intensify the membrane damage. This in turn allows more D−AgNCs to enter the bacteria and generate localized high ROS concentration within the cell and subsequently induce a severe DNA damage, leading to their high killing effect over bacteria.

observe any increase in the ROS production on the daptomycin-treated group. This is expected as the daptomycin’s disruption of the bacterial membrane is not facilitated through the ROS production but by the insertion and interaction of its lipophilic tail with the lipid layer on the membrane. In comparison, AgNCs, D+AgNCs, and D−AgNCs groups showed an elevated ROS production when compared to the untreated and daptomycin controls. The incorporation of NAC in the treatment groups could eliminate the AgNCs-induced intracellular ROS production (Figure 4), bringing down the intracellular ROS close to the basal level. This further supports our previous observation that the ROS induced by AgNCs was involved in damaging the cell membrane (Figure 2a,b) and DNA (Figure 3d). It is possible that this intracellular ROS was originated from the intracellular release of Ag(0) from the AgNCs. Owing to the basal level of ROS produced by the cells as respiratory product, the released Ag(0) would be oxidized to form Ag+, further propagating the ROS production and inducing injury to the subcellular components that could kill the bacteria.24,31 Comparison between the treatment groups showed that D− AgNCs induced more intracellular ROS when compared to the AgNCs. This is in good agreement with our previous observations, in which the D−AgNCs have been demonstrated to produce more damage on the cell membrane that allow more

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: sodium borohydride (NaBH4), L-glutathione reduced (GSH), N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimide hydrochloride (C8H17N3·HCl, EDC), N-hydroxysuccinimide (C4H5NO3, NHS), agar, 2′,7′-dichlorofluorescein diacetate (DCFH-DA), propidium iodide (PI), lysozyme, lysostaphin, paraformaldehyde (PFA), 7940

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ACS Nano GenElute Bacterial Genomic DNA Kits, Triton X-100, N-acetyl-Lcysteine (NAC), and synthesized oligonucleotides strands were purchased from Sigma-Aldrich; silver nitrate (AgNO3) and sodium hydroxide (NaOH) were purchased from Merck; Luria−Bertani (LB) was obtained from Becton Dickinson; Hoechst 33342, Click-iT TUNEL Alexa Fluor 488 Imaging Assay, and ProLong Gold antifade reagent with DAPI were purchased from Life Technologies, and the lipid peroxidation (MDA) assay kit was purchased from Abcam. The Amplex red hydrogen peroxide/peroxidase assay kit was purchased from Invitrogen. Daptomycin from Sigma was provided by Dr. Lim Tze Peng (Singapore General Hospital (SGH), Singapore). S. aureus (ATCC 25923) was a kind gift from Prof. Tan Kai Soo (Faculty of Dentistry, National University of Singapore, NUS). Instruments. A PD-10 desalting column (GE Healthcare UK Ltd.) containing 8.3 mL of Sephadex G-25 medium with a molecular weight exclusion limit of 10 kDa was used to purify AgNCs after synthesis. Inductively coupled plasma optical emission spectrometry (ICP-OES) was used to determine the concentration of AgNCs in solution, which was measured through iCAP 6000 Series, Thermo Scientific. UV−vis absorption and photoluminescence (PL) spectra were recorded by a Shimadzu UV-1800 photospectrometer and a PerkinElmer LS-5S fluorescence spectrometer, respectively. Fourier transform infrared spectra (FTIR) were measured through a Bio-Rad FTS 3500 spectroscopy. Thermogravimetric analysis (TGA) was conducted on a Shimadzu DTG-60AH analyzer under N2 atmosphere. FTIR-TGA was tested on Shimadzu IR Prestige-21 and DTG-60A instruments under N2 atmosphere. Hydrodynamic size distribution 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 microplate reader Tecan Infinite M200 Pro. The bacteria fluorescent images were taken with an epifluorescence microscope Leica DMI6000. Native polyacrylamide gel electrophoresis (PAGE) was carried out on a Bio-Rad mini-protean tetra cell system, and the gel was viewed with the fluorescent/visible imaging Syngene G:BOX F3 gel-imaging unit. Synthesis of AgNCs and D−AgNCs. Preparation of AgNCs. 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. Freshly prepared aqueous solutions of GSH (150 μL, 50 mM) and AgNO3 (125 μL, 20 mM) were mixed in ultrapure water (4.85 mL) under vigorous stirring at room temperature. After that, 50 μL of the as-prepared NaBH4 solution (112 mM) was added drop by drop, and the solution turned to deep red after ∼5 min reaction. The solution was incubated without stirring at room temperature for ∼4 h, and it gradually turned to colorless as the decomposition of AgNCs occurred through the etching by the thiolate ligands and oxygen. After that, 50 μL of the asprepared NaBH4 solution (112 mM) was added drop by drop into the colorless solution under vigorous stirring, and the solution turned light brown after ∼30 min. Finally, the solution was incubated without stirring at room temperature for ∼8 h to obtain the final AgNCs product with strong red emission. Preparation of D−AgNCs. The as-prepared AgNCs were first purified via a PD-10 desalting column. Aqueous solutions of daptomycin (2 mM), EDC (400 mM), and NHS (200 mM) were freshly prepared. In a typical synthesis of D−AgNCs, the purified AgNCs (5 mL) were first mixed with daptomycin (125 μL, 2 mM), followed by the addition of EDC (100 μL, 400 mM) and NHS (100 μL, 200 mM) in the solution. The mixed solution was reacted for ∼1 h under vigorous stirring at room temperature. The as-synthesized antibiotic-conjugated AgNCs (D−AgNCs) were then purified by running through a PD-10 desalting column, followed by a rotary evaporator treatment to concentrate the sample. The final product was ∼1 mM (on the basis of Ag atoms), which was determined by ICPOES.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.6b03862. Experimental procedures bacterial-related tests like ROS measurement, bacterial membrane damage, and DNA damage; TEM images; molecular structure of daptomycin; FTIR spectra; TGA−FTIR analysis; TGA analysis; relative bacterial growth, etc. (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was financially supported by the Ministry of Education (MOE), Singapore, under Grant No. R-279-000481-112 and SingHealth Foundation SHF/FG511P/2012. K.Z. thanks the National University of Singapore for her research scholarship. We thank Prof Tan Kai Soo (Faculty of Dentistry, NUS) for her kind gift of S. aureus. REFERENCES (1) Costerton, J. W.; Stewart, P. S.; Greenberg, E. P. Bacterial Biofilms: A Common Cause of Persistent Infections. Science 1999, 284, 1318−1322. (2) Rai, M.; Yadav, A.; Gade, A. Silver Nanoparticles as A New Generation of Antimicrobials. Biotechnol. Adv. 2009, 27, 76−83. (3) Stewart, P. S.; Costerton, J. W. Antibiotic Resistance of Bacteria in Biofilms. Lancet 2001, 358, 135−138. (4) Mah, T. F. C.; O’Toole, G. A. Mechanisms of Biofilm Resistance to Antimicrobial Agents. Trends Microbiol. 2001, 9, 34−39. (5) Taubes, G. The Bacteria Fight Back. Science 2008, 321, 356−361. (6) Neu, H. C. The Crisis in Antibiotic Resistance. Science 1992, 257, 1064−1073. (7) Cohen, M. L. Epidemiology of Drug Resistance: Implications for A Post-Antimicrobial Era. Science 1992, 257, 1050−1055. (8) Hancock, R. E. W.; Sahl, H. G. Antimicrobial and Host-Defense Peptides as New Anti-Infective Therapeutic Strategies. Nat. Biotechnol. 2006, 24, 1551−1557. (9) Lemire, J. A.; Harrison, J. J.; Turner, R. J. Antimicrobial Activity of Metals: Mechanisms, Molecular Targets and Applications. Nat. Rev. Microbiol. 2013, 11, 371−384. (10) Steenbergen, J. N.; Alder, J.; Thorne, G. M.; Tally, F. P. Daptomycin: A Lipopeptide Antibiotic for the Treatment of Serious Gram-Positive Infections. J. Antimicrob. Chemother. 2005, 55, 283− 288. (11) Muangsiri, W.; Kirsch, L. E. The Protein-Binding and Drug Release Properties of Macromolecular Conjugates Containing Daptomycin and Dextran. Int. J. Pharm. 2006, 315, 30−43. (12) Vilhena, C.; Bettencourt, A. Daptomycin: A Review of Properties, Clinical Use, Drug Delivery and Resistance. Mini-Rev. Med. Chem. 2012, 12, 202−209. (13) Nikaido, H. Multidrug Resistance in Bacteria. Annu. Rev. Biochem. 2009, 78, 119. (14) Levy, S. B.; Marshall, B. Antibacterial Resistance Worldwide: Causes, Challenges and Responses. Nat. Med. 2004, 10, S122−S129. (15) Wu, Y. L.; Scott, E. M.; Po, A. L. W.; Tariq, V. N. Ability of Azlocillin and Tobramycin in Combination to Delay or Prevent Resistance Development in Pseudomonas Aeruginosa. J. Antimicrob. Chemother. 1999, 44, 389−392. 7941

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DOI: 10.1021/acsnano.6b03862 ACS Nano 2016, 10, 7934−7942