Mercaptopyrimidine-Conjugated Gold Nanoclusters as

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Mercaptopyrimidine-Conjugated Gold Nanoclusters as Nanoantibiotics for Combating Multidrug-Resistant Superbugs Youkun Zheng, Weiwei Liu, Zhaojian Qin, Yun Chen, Hui Jiang, and Xuemei Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00452 • Publication Date (Web): 31 Jul 2018 Downloaded from http://pubs.acs.org on August 2, 2018

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Bioconjugate Chemistry

Mercaptopyrimidine-Conjugated

Gold

Nanoclusters

as

Nanoantibiotics for Combating Multidrug-Resistant Superbugs Youkun Zheng†, Weiwei Liu†, Zhaojian Qin, Yun Chen, Hui Jiang*, Xuemei Wang*

State Key Laboratory of Bioelectronics, National Demonstration Center for Experimental Biomedical Engineering Education, School of Biological Science and Medical Engineering, Southeast University, Nanjing 210096, China

ABSTRACT: Widespread bacterial resistance induced by the abuse of antibiotics eagerly needs the exploitation of novel antimicrobial agents and strategies. Gold nanoclusters (Au NCs) have recently emerged as an innovative nanomedicine, but study on their antibacterial properties especially towards multidrug resistant (MDR) bacteria is scarce. Herein, we demonstrate that a novel class of Au NCs, mercaptopyrimidine conjugated Au NCs, can act as potent nanoantibiotics targeting these intractable superbugs in vitro and in vivo, without induction of bacterial antibiotic resistance and noticeable cytotoxicity to mammalian cells. The Au NCs kill these superbugs through a combined mechanism including cell membrane destruction, DNA damage, and reactive oxygen species (ROS) generation, and exhibit excellent treatment effects in both macrophages and animal infection models induced by methicillin-resistant Staphylococcus aureus as representative. Moreover, the induction of intracellular ROS production in bacterial cells mainly attributed to the Au NCs’ intrinsic oxidase- and peroxidase-like catalytic activities has been demonstrated for the first time.

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INTRODUCTION Antibiotics have made great contribution to rescuing human life in the past several decades. However, the abuse of antibiotics has led to the recent emergence of a great deal of multidrug resistant (MDR) bacterial strains, including the most notorious “ESKAPE” superbugs (i.e., MDR Acinetobacter baumannii, MDR Pseudomonas aeruginosa, MDR Klebsiella pneumoniae, MDR Enterobacter

species,

methicillin-resistant

Staphylococcus

aureus

(MRSA),

and

vancomycin-resistant Enterococcus faecium (VRE)), which have become one of the most serious global public health crises.1,2 It has been attempted to reverse the bacterial resistance to antibiotics by exploiting new antimicrobial drugs and chemically modifying extant antibiotics.3,4 Unfortunately, the pace of antibiotic development cannot catch up with the development of bacterial resistance, and the increasing rates of MDR also invalidate the utility of even the most potent antibiotics.5 Therefore, it is urgently needed to develop new antimicrobial approaches especially non-antibiotic drugs to fight with superbug infections. Based on high specific surface area and exceptional antimicrobial performance (either intrinsic or chemically incorporated), engineered nanostructures have been widely studied as one of the most promising antimicrobial substances, while the microbe may not be able to develop resistance to them.6−9 For example, metals and metal oxides nanomaterials which possess excellent antibacterial activity, have been used as potential agents to combat MDR bacterial infections.10−13 However, although elemental nanoparticles (NPs), such as Ag NPs, have an intrinsic antimicrobial capacity, they also raise a severe safety menace for practical application due to their cytotoxicity to human body.14,15 Therefore, it is necessary to find alternative antibacterial candidates with proper human safety to insure the clinical transformation. In this regard, the “noblest” metals-gold, has a greater advantage over silver due to its bioinertia and high stability. Nanostructures based on this element have also been extensively proven to possess good biocompatibility in animal system.16,17 This biocompatibility still remains even if the size is further reduced to nanoclusters (NCs) range.18 Interestingly, many Au NPs have also been used as antibacterial materials after they have been grafted with known antibacterial compounds, such as small molecule antibiotics,19,20 antimicrobial peptides,21,22 and cationic ligands,23 on their surface, presumably using Au nanostructures as driven antibiotic carriers. Unlike large Au NPs, smaller Au NCs have recently emerged as innovative nanomedicine with interesting 2


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physicochemical properties, such as size-dependent fluorescence and catalytic activity.24−26 A recent research has demonstrated that ultrasmall Au NCs (~ 2 nm) exhibit antibacterial activity through inducing intracellular reactive oxygen species (ROS) production, whereas larger Au nanoparticles (~ 6 nm) are incapable.27 However, studies on the impact of Au NCs on superbugs are lacking, whether in vitro or in vivo. Herein, we synthesized Au NCs conjugated mercaptopyrimidine as nanoantibiotics for combating

ESKAPE

superbugs.

4-amino-2-mercaptopyrimidine

Four

(AMP),

mercaptopyrimidine

analogues,

4,6-diamino-2-mercaptopyrimidine

namely, (DAMP),

4-amino-6-hydroxyl-2-mercaptopyrimidine (AHMP), and 4,6-dihydroxyl-2-mercaptopyrimidine (DHMP), were used as ligands for the synthesis of Au NCs. These ligands have been also used in the synthesis of Au NPs in the previous study.10 Unlike mercaptopyrimidine-capped Au NPs, the as-synthesized Au NCs especially AuDAMP have superior antibacterial performances against both Gram-positive and Gram-negative bacteria. The antibacterial properties of AuDAMP are mainly attributed to their oxidase- and peroxidase-like activities. In contrast, large-sized Au NPs exhibit relatively weak antibacterial activity due to their weak mimic enzyme activity, particularly against Gram-positive bacteria. In addition, the Au NCs exhibit detectable cytotoxicity and hemolysis for mammal cells but have excellent therapeutic effects against superbug’s infections, as demonstrated through macrophages infection model as well as mice infection models. RESULTS AND DISCUSSION We firstly synthesized mercaptopyrimidine-conjugated Au NCs via reduction of gold precursor (HAuCl4) by relying on the ligand's inherent reducibility in the presence of the mercaptopyrimidine ligands in 50% ethanol aqueous solution. The transmission electron microscopy (TEM) images show the sizes of ultrasmall AuAMP, AuDAMP, AuAHMP, and AuDHMP NCs are less than 2 nm. The zeta potential measurements indicate that amino-mercaptopyrimidine-conjugated Au NCs (AuDAMP, AuAMP, and AuAHMP) are positively charged, and hydroxyl-mercaptopyrimidine-conjugated Au NCs (AuDHMP) are negatively charged (Figure 1).

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Figure 1. Characterization of mercaptopyrimidine-conjugated Au NCs. (a) TEM images of AuDAMP, AuAMP, AuAHMP and AuDHMP. (b) Chemical structures of the corresponding mercaptopyrimidine molecules. (c) Size distribution and (d) zeta potential of Au NCs. Then we investigated the antibacterial activities of mercaptopyrimidine-conjugated Au NCs by determination of minimal inhibitory concentration (MIC) using a broth-dilution method.28,29 Firstly, we select two antibiotic-sensitive strains Escherichia coli ATCC35218 (Gram-negative bacteria) and S. aureus ATCC29213 (Gram-positive bacteria) as bacterial model to test the antimicrobial abilities of these Au NCs. AuAMP, AuDAMP, AuAHMP, and AuDHMP are active against two representative strains, with a MIC ranging from 2 µg/mL to 32 µg/mL directly (Table 1), suggesting that mercaptopyrimidine-conjugated Au NCs may have a broad spectrum of antibacterial properties. The antimicrobial ability of AuDAMP is much better than that of AuAMP, AuAHMP,

and

AuDHMP.

Furthermore,

we

also

assess

the

MIC

of

these

mercaptopyrimidine-conjugated Au NCs against clinically isolated ESKAPE strains, i.e., MDR E. coli, MDR A. baumannii, MDR P. aeruginosa, MRSA, MDR K. pneumoniae, and VRE. These superbugs are isolated from the sputum samples of clinical patients with respiratory tract infections. The resistance of ESKAPE is shown in Table S1. MDR Gram-negative strain, MDR A. baumannii was resistant to 15 common antibiotics and just susceptible to aminoglycosides and tetracyclines. MDR E. coli, MDR K. pneumonia, and MDR P. aeruginosa were resistant to 14, 12, and 10 antibiotics, respectively. MDR Gram-positive strains, MRSA and VRE, were resistant to 4


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more than 10 antibiotics. Moreover, three classes of conventional antibotics were used as control, which are levofloxacin (a type of quinolones, broad spectrum), vancomycin (a type of glycopeptides, the first-line drug for the treatment of MRSA infections), and colistin (a type of polymyxins, to treat Gram-negative bacteria). Their antibacterial effects are not satisfying. ESKAPE were all resistant to levofloxacin. As a typical superbug’s therapeutic agent, colistin was also found to be inactive against MDR A. baumannii (Table 1, Table S1). In contrast, we found that AuDAMP can inhibit the multiplication of MDR E. coli, MDR A. baumannii, MDR P. aeruginosa, MDR K. pneumoniae, MRSA, and VRE, with a MIC of 4 µg/mL, 2 µg/mL, 4 µg/mL, 2 µg/mL, 2 µg/mL, and 8 µg/mL, respectively (Table 1). By contrast, AuAMP, AuAHMP, and AuDHMP show relatively weak antibacterial properties against clinical ESKAPE groups. In particular, AuDHMP show virtually inactive against VRE. The lower MIC values of AuDAMP indicate

that

their

antimicrobial

capacity

is

superior

to

that

of

other

mercaptopyrimidine-conjugated Au NCs. In addition, AuDAMP had a red-emitted fluorescence (620 nm) with a longer fluorescence lifetime (Figure S1), and almost no agglomeration or fluorescence quenching occurs during long-term storage in aqueous solution (Figure S2). Considering that AuDAMP have excellent stability and show the superior antibacterial characteristic even better than conventional antibiotics, we used the NCs in the following studies. To compare the size effect of particles on the antimicrobial activity, we also tested the antibacterial properties of DAMP-Au NPs with larger size (~ 6 nm) via synthesis by NaBH4 reduction (Figure S3). Compared to AuDAMP NCs, AuDAMP NPs have relatively weak antibacterial activity, particularly against Gram-positive bacteria (Table 1). Considering the difference in surface ligand density caused by the different sizes of Au NPs and Au NCs, we will also compare the antimicrobial properties of the two kinds of Au nanostructures based on the surface DAMP concentrations. The elemental analysis results show that the Au-to-DAMP molar ratios in Au NPs and Au NCs are 1:1 and 1:1.4, respectively. The MIC data based on the same DAMP concentrations also show that the Au NCs have better antibacterial activities (Table S2). This result is different with previous reports on 6-mercaptohexanoic acid stabilized Au NPs,27 indicating that in addition to the size effect, ligands might also significantly affect the antibacterial properties of nanostructures (Table 1).30 In fact, DAMP is an analogue of 2-mercaptopyrimidine in E. coli tRNA, and although it has almost no direct pharmacological action in itself (Table 1), it is 5



usually used as a pharmaceutical intermediate for the synthesis of antimicrobial agents.31 Previous report has also shown that DAMP-Au NPs can kill some Gram-negative bacteria but inactive against Gram-positive bacteria.10 Furthermore, we performed the killing kinetic assays to determine the changes in the antibacterial agent (i.e. AuDAMP) activity over the time. As illustrated in Figure S4, AuDAMP show a concentration- and time-dependent antibacterial behavior against test bacteria, with different dynamics for different strains. In particular, AuDAMP caused a markedly decrease in the amount of S. aureus ATCC29213 and MRSA colonies within 6 h and all bacterial cells were eradicated in 12 h at 2× MIC concentration (f and g of Figure S4). These data show that the AuDAMP NCs possess fast killing kinetics, besides highly antibacterial activities against superbugs. Table 1. Antibacterial activities indicated with MIC (µg/mL).

Note: DAMPAu(I) is the precursor complex during the synthesis of Au NCs.

Bacterial biofilm is a main source of persistent infections and can enhance drug resistance.29,32 Thus, we also evaluated the effects of AuDAMP on biofilm formation and mature biofilms using MRSA as a model. Remarkably, AuDAMP has a highly effective anti-biofilm activity, and very low concentrations of relevant Au NCs can significantly inhibit the formation of MRSA biofilms (Figure S5). This result is similar to our previous report on inhibition of the biofilm formation of MRSA by organometallics,29 indicating the excellent anti-biofilm formation features by AuDAMP. In addition, AuDAMP can also eliminate mature biofilms of MRSA at lower concentrations (Figure S6), whereas most known antibacterial agents, including noble metal nanoparticles, cannot exhibit similar effect.33 Long-term bacterial resistance is one of the biggest challenges in the current therapy of infections. The use of any new antibiotics, without exception, led to the emergence of 6


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drug-resistant strains,5 and even bacteria that tolerate Ag NPs have emerged.34 To evaluate the potential resistance of bacteria to Au NCs, standard strain S. aureus ATCC29213 were repeatedly exposed to AuDAMP at a sub-lethal concentration (one third of MIC). As expected, S. aureus ATCC29213 rapidly developed significant resistance to conventional antibiotic vancomycin at sublethal dose within a few days. However, no resistance development towards the AuDAMP was demonstrated even after continuous passages for thirty days (Figure 2). Indeed, it is well known that vancomycin-resistant S. aureus is one of severe threat to public health.35,36 In contrast, there is no evidence that bacteria can resistant to antibacterial Au nanomaterials.23,37,38 These results all indicate that AuDAMP are a novel and effective nanoantibiotics against superbugs and do not cause drug tolerance.

Figure 2. Development of drug resistance towards AuDAMP and vancomycin against S. aureus ATCC29213 upon multiple sublethal dose exposures. The antimicrobial mechanism of the Au NCs was visited using MDR E. coli and MRSA as models. The high positive charge on the surface of AuDAMP can promote electrostatic adsorption onto the bacterial surface, enabling easy and efficient internalization (Figure S7). To study whether AuDAMP destroy bacterial cell membranes, we performed the bacterial life and death identification experiments using our previous method.29 Figure 3a shows the confocal laser scanning microscopy (CLSM) images of MDR E. coli and MRSA treated with AuDAMP. The control group bacteria show only green fluorescence, demonstrating that the bacterial cells are 7



living. However, after treated with AuDAMP (final concentration, 20 µg/mL), almost all bacterial cells emit strong red fluorescence, which shows negligible emission by the green fluorescence channel, indicating that the bacterial cell membranes have been completely disrupted and superbugs are killed.

Figure 3. (a) CLSM images of MDR E. coli and MRSA untreated and treated with AuDAMP NCs (the final concentration, 20 µg/mL) at 37 °C for 2 h, respectively. The dead cells were visualized by PI staining (red), while the SYTO 9 (green) helped to identify all cells. (b) SEM micrographs of MDR E. coli (top) and MRSA (bottom) untreated or treated with AuDAMP. Scale bars = 2 µm. (c) Agarose gel electrophoresis result of the extracted genomic DNA of MDR E. coli and MRSA untreated and treated DAMPAu(I), AuDAMP NCs, and AuDAMP NPs, respectively. (d) Relative ROS yield by normalizing the ROS level after incubation for 2 h, where the ROS values of the 8


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control group was defined as 1. To further confirm the antibacterial mechanism of AuDAMP, the scanning electron microscope (SEM) study was carried out. The untreated MDR E. coli and MRSA cells show intact and smooth morphology (Figure 3b). After treatment with 20 µg/mL of AuDAMP at 37 °C for 2 h, the MDR E. coli and MRSA cells change into a wizened and damaged state. Moreover, for most of the pathogenic cells especially MDR E. coli, cellular contents leakage can be observed (Figure 3b). These results confirm that AuDAMP can strongly interact with the pathogenic cell membranes which are not only Gram-positive but also Gram-negative and readily kill such superbugs by cell damage. Following the destruction of comprehensive cell membrane, the Au NCs can easily enter the bacteria and interact directly or indirectly with the bacterial genomic DNA, thereby inducing the DNA destruction. To confirm this, we extracted and electrophoretically analyzed the genomic DNA of bacteria after treatment with Au DAMP, as presented in Figure 3c. The untreated and DAMPAu(I)-treated MRSA shows a compact DNA band, indicating there is no DNA destruction. However, the Au NCs and Au NPs treated group showed a remarkable smearing on its DNA band, suggesting the presence of shorter DNA fragments and genomic DNA destruction caused by the treatment. Many nanoparticles-based antibacterial agents, including Au NCs, attribute their efficiency to their ability to modulate the ROS production that ultimately eliminates the bacteria.27,37,39 As such, we called on whether the antibacterial efficiency of AuDAMP was driven by ROS generation. As illustrated in Figure 3d, AuDAMP treatment induces a dramatic increase in the intracellular ROS production in bacteria when compared to the water-treated group. Consistent with previous reports,10,27 mercaptopyrimidine-stabilized Au NPs do not induce any visible increase of ROS in bacterial cells (Figure 3d). When antioxidant N-acetyl-L-cysteine (NAC) was incorporated, the ROS production induced by Au NCs was observably eliminated, pulling down the ROS to the original level (Figure 3d). These results clearly demonstrate that intracellular ROS production was the key factor in the potent antibacterial capability of AuDAMP. Based on the above observations, we further studied the reason why AuDAMP can induce intracellular ROS generation and Au NPs cannot. This result allows us to accurately identify the fundamental mechanism that leads to the ROS production and the subsequent antibacterial ability. 9



It is noteworthy that previous studies have found that the ROS-induced antibacterial performances of noble metal-based nanoantibiotics are generally attributed to their intrinsic oxidase-like and peroxidase-like catalytic capacity.12,40−42 And considering that the size of the Au NPs significantly affects its catalytic performance,26,43 we therefore hypothesize that Au NCs can induce ROS generation possibly because of their oxidase-like and peroxidase-like natures, whereas the Au NPs cannot. To explore the oxidase-like nature of Au nanoantibiotics, we employed the 3,3′,5,5′-tetramethylbenzidine (TMB) as probe, which appears a maximum absorbance at 653 nm when it is oxidized (producing a blue product). The results show a time-dependent increase in TMB oxidation catalyzed by Au NCs (Figure 4a). However, there is no obvious oxidized TMB characteristic absorbance after the addition of Au NPs, even after 30 minutes (Figure S8). To further confirm the oxidase-like property of the Au nanoantibiotics, we detected the generation of hydrogen peroxide (H2O2) in the catalytic oxidation process of ascorbic acid (AA, an effective antioxidant in cells) by the Au nanoantibiotics. As shown in Figure 4b, a remarkable increase of H2O2 is detected in the presence of Au NCs compared with control group (AA only), while there is almost no increase in the presence of large-size Au NPs. These data unarguably affirm that the Au NCs can catalyze the oxidation of antioxidants and accompany H2O2 generation. We then evaluated the peroxidase-like performance of the Au nanoantibiotics by following the catalysis of the TMB by H2O2. The results from the UV-vis absorption spectra suggest that the relevant Au NCs can catalyze the oxidation of TMB by H2O2 (Figure 4c), as the expected one-electron oxidation intermediate product TMB+ was detected (inset in Figure 4c),44 which in turn is a symbol of the Au NC’s peroxidase-like activity. The catalytic oxidation rates of these Au NCs increase with increasing concentrations and reaction times (Figure S9). Similarly, the Au nanoantibiotics also exhibit a highly size-dependent peroxidase-like performance because the large-size Au NPs have almost no catalytic activity (Figure S10). Despite the specific mechanism of the mimic peroxidase catalysis is not yet clear, one possible reason for the peroxidase-like nature of the Au NCs might originate from the Au NC’s ability to decompose H2O2 species to produce hydroxyl radicals (•OH). To further understand the reason why the Au NCs have a better catalytic property than the Au NPs, we therefore compared their productivities in producing •OH from H2O2 using terephthalic acid (TA) as a molecular probe, which can easily combine •OH to form fluorescent substance 2-hydroxy-TA (inset in Figure 4d).44 10


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As illustrated in Figure 4d, that strong fluorescent was observed in the systems containing TA, Au nanoantibiotics, and H2O2, whereas only a very feeble fluorescent was observed in the absence of either Au nanoantibiotics or H2O2. This finding indicated that the •OH species are mainly generated from the decomposition of H2O2 in the presence of Au nanoantibiotics as catalysts. Notably, the fluorescent intensity of the system of H2O2, Au NCs, and TA is much stronger than that of the system of H2O2, Au NPs, and TA. This data demonstrates that the Au NCs are more efficient in the decomposition of H2O2 to generate •OH than large-size Au NPs, which may be related to their better catalytic performance toward the oxidation of TMB by H2O2 relative to Au NCs. In general, the data from both membrane integrity assay and SEM experiments uncover that AuDAMP can effectively damage the bacterial cell membranes and change their permeability. The change in cell membrane permeability then allows AuDAMP to easily internalize into bacterial cells, inducing the destruction of bacterial genomic DNA. More importantly, due to the intrinsic oxidases- and peroxidase-like catalytic properties, AuDAMP could strongly induce intracellular ROS production after their entry. The generation of intracellular ROS further accelerates the death of bacteria. Due to the fact that bacteria are difficult to develop resistance to antimicrobial agents that cause membrane damage,29,45 the damage ability of bacterial cell membranes also enables AuDAMP to effectively against drug resistance.

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Figure 4. Oxidase- and peroxidase-like properties of the Au nanoantibiotics. (a) Time-dependent absorbance spectra of the TMB catalyzed by Au NCs with the characteristic absorption maximum of oxidized TMB at 653 nm. (b) The concentration of H2O2 generated in the catalytic system: AA alone and AA with the different Au nanoantibiotics. The data indicate the means and standard deviation (SD) from three parallel experiments. P-values were calculated by the Student’s test: **p < 0.01. (c) Au NCs+H2O2+TMB, where the maximum absorbance values for the TMB+ intermediate (responsible for the characteristic blue color) are at 370 nm and 653 nm. The inserted images (tubes) represent the visual color changes of TMB in different reaction systems from left to right: H2O2+TMB; Au NCs+TMB; Au NCs+H2O2+TMB. (d) The fluorescence spectra for detection of hydroxyl radicals (•OH) from the different reaction systems. Here, the intermediate fluorescence product, 2-hydroxy-TA, presents a maximum emission wavelength at 435 nm. To evaluate the preliminary biocompatibility of these Au NCs toward mammalian cells, we performed MTT assay by using human normal liver L02 cells and normal lung AT II cells as mammalian cells models. As illustrated in Figure S11a, the AuDAMP show negligible toxicity to L02 and AT II cells at dose of up to 256 µg/mL, which is much higher than the MIC for bacteria (Table 1). Meanwhile, the hemolytic behavior was also investigated by treatment of human erythrocytes with AuDAMP. Similar to the results of cytotoxicity experiments, no noticeable hemolysis was observed for AuDAMP at a relatively higher dose (Figure S11b). In addition, the in vivo cytotoxicity was also evaluated. The histopathological results showed no observable damage in the main organs of mice between the AuDAMP-treated group and untreated group (Figure S11c). Overall, AuDAMP exhibit excellent biocompatibility to both mammalian cells and living body, and may be suitable for further in vivo applications. To further reveal the potential of biocompatible AuDAMP for the in vivo applications, the in vitro antibacterial capacity was also evaluated with co-culture model of bacteria (i.e. MRSA) and macrophages (i.e. RAW 264.7), a conventional mononuclear phagocyte system infection model.45 MRSA were marked with SYTO 9 dye before co-culture with RAW 264.7 macrophages. Comparing the untreated group with the DAMPAu(I)-treated group, the number of intracellular MRSA significantly decreased in the presence of AuDAMP (Figure 5a). The amount of MRSA colonies further testified the high antibacterial property of AuDAMP toward MRSA inside the macrophage cells (Figure 5b). In the meantime, the cell nuclei of macrophages (blue stained by 12


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Hoechst 33342 dye) remain intact after treatment with DAMP and AuDAMP, indicating that the RAW 264.7 cells are still alive. These results clearly demonstrate that AuDAMP can kill superbugs inside the macrophages with high efficiency, but with no harm to the macrophages due to excellent biocompatibility.

Figure 5. (a) CLSM images of MRSA-infected RAW 264.7 macrophages untreated and treated by DAMPAu(I) and AuDAMP NCs for 2 h. MRSA were marked by green fluorescent SYTO 9 dye and the nuclei of RAW 264.7 cells were stained with blue fluorescent Hoechst 33342 dye. (b) Photographs and corresponding statistics of MRSA colonies formed on LB-agar plates after co-culture experiments of MRSA and RAW 264.7 macrophages. To assess the application of AuDAMP to fight superbugs in vivo, we first used AuDAMP in a mice skin infection model. MRSA (109 CFUs/wound) were selected to infect the wounds, which was followed by single-dose treatment of AuDAMP (0.1 mL, 10 µg/mL). Vancomycin (0.1 mL, 10 µg/mL) and normal saline (NaCl, 0.9%) were employed for assaying as positive control and negative control, respectively. Treatment effect was evaluated by measuring the wound sizes every 2 days and by calculating the colonies counts for the tissue homogenate of the infected sites on day 10 (Figure 6). After 10 d treatment, the wounds of mice are basically healed in the AuDAMP 13



treatment group and the vancomycin treatment group, while the relative wound size of the negative control group was still as high as about 35% (a and b of Figure 6). Considering the whole healing process, AuDAMP as well as vancomycin treatment can expedite wound healing and reduce the probability of infection and death. Indeed, after normal saline treatment, quantification of the bacterial burden in the wound showed 6.3×106 CFU/g of MRSA. Mice treated with AuDAMP showed a bacterial burden of 3.4×104 CFU/g, which was a noteworthy reduction compared to the normal saline-treated group (with 99% bactericidal efficacy) (Figure 6c). In addition, a mice pneumonia model was also used to further evaluate the treatment effect of AuDAMP (Figure S12). In the normal saline-treated group, all mice died after 5 days of treatment, and the amount of MRSA in the lung was as high as 107 CFU/g level. Compared with normal saline-treated group, all mice survived after 6 days of AuDAMP therapy, and the amount of MRSA in the lung was decreased significantly by an order of magnitude. These results all show that the as-prepared Au NCs have excellent therapeutic effect that are comparable to traditional antibiotics, and have great potential for clinical application of MDR superbug’s infections.

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Figure 6. The therapeutic effect of AuDAMP on a mice skin infection model. (a) Representative photographs of the MRSA-infected wound untreated and treated with AuDAMP (0.1 mL, 10 µg/mL). (b) Their corresponding wound sizes (relative area versus initial area). (c) Photographs and the corresponding statistical histogram of bacterial colonies formed on the LB-agar plates derived from the homogenized tissue dispersions of the infected sites of mice injected with AuDAMP (10 d). Normal saline (NaCl, 0.9%) and vancomycin (0.1 mL, 10 µg/mL) were used for testing as negative control and positive control, respectively. Error bars represent the standard deviation of three repeated measurements. CONCLUSION In summary, a new class of Au NCs stabilized with selected mercaptopyrimidine analogues ligands has been shown to be superior nanoantibiotics against both MDR Gram-negative and Gram-positive superbugs in vitro and in vivo, without inducing drug resistance and showing noticeable cytotoxicity and hemolysis to mammalian cells. The broad spectrum antibacterial property is attributed to the Au NCs’ oxidase-like and peroxidase-like catalytic activities has been demonstrated for the first time. When the enzymes-like Au NCs were internalized, this intrinsic catalytic performance could induce an increase of intracellular ROS generation, conferring bactericidal efficacy as the end result. The present study not only extends the versatility of nanomaterial-based antibiotics, but also offers a new perspective for biomedical applications of ultrasmall Au NCs. EXPERIMENTAL SECTION Synthesis and Characterization of AuAMP, AuDAMP, AuAHMP, and AuDHMP. The preparation of Au NCs was described in our previous study.46 For typical synthesis of mercaptopyrimidine-stabilized Au NCs, 2 mL of mercaptopyrimidine (10 mM, dissolved in 50% ethanol solution) and 1 mL of HAuCl4 (10 mM) were added to 7 mL of ultrapure water. The mixed solution was heated to 70 °C and continually reacted under gentle stirring (300 rpm) for 12 h. The synthesized Au NCs were purified using ultrafiltration and centrifugation to remove bulk gold and free mercaptopyrimidine.47 The purified Au NCs solution were stored at 4 °C in dark for further characterization. We synthesize AuAMP, AuAHMP, and AuDHMP using the same method. The morphologies of Au NCs were observed through transmission electron microscope (TEM, JEM-2100, Japan). The zeta potential was measured by Nano ZS Zetasizer 90 (Malvern, UK). 15



Antibacterial Activity Measurement. For evaluation of antimicrobial activity of Au NCs, E. coli ATCC35218, MDR E. coli, MDR A. baumannii, MDR P. aeruginosa, MDR K. pneumoniae, S. aureus ATCC29213, MRSA, and VRE were selected for use in this study. The MIC was determined using a broth dilution method.28 Bacteria (~1.0×107 CFU/mL) were cultured into LB medium of 2 mL supplemented with the Au NCs, antibiotics, or other agents. The final concentrations range from 0.5 to 128 µg/mL. The as-prepared bacterial solutions were inoculated at 37 °C for 24 h. The viable colony counts were determined before and after incubation. Macrophages Infection Model. MRSA infected RAW 264.7 macrophages were built as an infection model to evaluate the antibacterial capacity of AuDAMP inside the macrophages. For visualization, MRSA (~1.0×108 CFU/mL) were marked with SYTO 9 dye though co-incubated with SYTO 9 for 30 min. Similar, the RAW 264.7 macrophage cells (~1.5×106 cells/mL) were stained with Hoechst nuclear dye for cell visualization. Then, Hoechst-labeled macrophages were subsequently with SYTO 9-labeled MRSA at a MOI (multiplicity of infection) of 10:1 and cultured for 2 h at 37 °C in medium without antibiotics. After cultured, the suspensions were centrifuged at 5000 rpm for 5 min, and the cells were washed with sterile normal saline for three times. Afterwards, RAW 264.7 cells were incubated with medium containing 10 µg/mL AuDAMP for another 2 h after washing for three times. The treatment of 10 µg/mL vancomycin was employed as a control. The mixtures were observed using a CLSM. To further quantify the efficacy, 100 µL diluted mixtures with and without the treatment of AuDAMP were then coated onto LB plates. The amount of bacterial colonies was computed after incubation. Mice Skin Infection Model. 4-week old BALB/c female mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). All animal experiments were performed according to the guidelines for Animal Experimentation with the approval of the National Institute of Biological Science and Animal Care Research Advisory Committee of Southeast University. To investigate the in vivo antibacterial capacity of Au NCs, the MRSA-infected mice model was prepared. 200 µL of MRSA (1.0×109 CFU/mL) in normal saline was subcutaneously injected into the mice. One hour after infection, the mice in the experimental group are intraperitoneally injected with Au NCs (0.1 mL, 10 µg/mL), the positive control group is injected with vancomycin (0.1 mL, 10 µg/mL), and the blank control group is injected with normal saline (NaCl, 0.9%). After 10 d of treatment, the bacterial amounts in the wounds were 16


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counted. Pneumonia Model. The pneumonia model was established by our previous method.48 Mice were intranasally infected with 100 µL suspension of MRSA (1.0×107 CFU/mL) in normal saline used dropwise to the nares. After administering Au NCs (0.1 mL, 10 µg/mL) for once a day, the mice were euthanized and the lung was harvested. The MRSA amounts in the lung samples were counted. ASSOCIATED CONTENT Supporting Information. Additional experimental section, such as bactericidal kinetic assay, biofilm formation inhibition test, CLSM observation of biofilm, mature biofilm elimination assay, membrane integrity test, bacterial morphological characterization, drug resistance development, ROS assay, mimic enzymes properties of Au Nanoantibiotics, MTT assay, hemolysis assay, and histopathology; fluorescence features, stability, TEM, time-kill curves, biofilm inhibition, UV-vis absorbance spectra, biocompatibility evaluation, and plate counting results (PDF). AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (H. J.) or [email protected] (X. W.) ORCID: 0000-0001-8044-758X (H. J.); 0000-0001-6882-7774 (X. W.) Author Contributions † These authors contributed equally to this work. Notes The authors declare no competing financial interest. ACKNOWLEDGMENTS This work was supported by National Key Research and Development Program of China (2017YFA0205300), the National Natural Science Foundation of China (91753106, 21675023, and 81325011), the Scientific Research Foundation of Graduate School of Southeast University (YBJJ1829), the Fundamental Research Funds for the Central Universities and Postgraduate Research & Practice Innovation Program of Jiangsu Province (KYCX17_0156).

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Table of Contents

Schematic for Au NCs with oxidase and peroxidase-like activities induce bacterial death through DNA damage, ROS generation and cell membrane damage.

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Mercaptopyrimidine-Conjugated Gold Nanoclusters as

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