Mercaptopyrimidine-Conjugated Gold Nanoclusters as. Nanoantibiotics for Combating Multidrug-Resistant Superbugs

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1 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 , China Corresponding author sungi@seu.edu.cn (H. J.); xuewang@seu.edu.cn (X. W.) S1

2 Supporting Information EXPERIMENTAL SECTION Bactericidal Kinetic Assay. In order to assess changes in the Au NCs activity and over time, the killing kinetic tests have been carried out. 1 Briefly, bacteria suspensions (~ CFU/mL) treated with different final concentrations of Au NCs at 37 C for 24 h on a shaking incubator (180 rpm), respectively. The viable colony counts were determined at different times during the incubate process. Tests were performed three times for each assay condition, and the average values were as the result records. Biofilm Formation Inhibition Test. MRSA were grown in LB for 12 h, diluted to CFU/mL in the absence or presence of Au NCs at different concentrations. Aliquots of 100 µl MRSA were transferred to sterile 96-well microtiter plate (5 wells per sample) and incubated at 37 C for 48 h. After incubation, the suspension was discarded, and plate was gently rinsed 3 times with sterile phosphate buffer saline (PBS), air dried, and dyed with 200 µl of 1% (w/v) crystal violet per well for 10 min. After rinsing the plate for 3 times with ultrapure water, the dye associated with attached biofilm cells was dissolved with 200 µl of ethanol. The absorbance at 595 nm of dye-ethanol solution was measured using a microplate reader (MK3, ThermoFisher). CLSM Observation of Biofilm. Confocal laser scanning microscopy (CLSM) assay was carried out according to a previously reported method. 2 MRSA incubated in LB medium was diluted to CFU/mL after overnight culture, and subsequently transferred into sterile 6-well plate (preloaded with coverslips), and incubated at 37 C for 48 h. After incubation, coverslips were gently rinsed 3 times with sterile PBS to rinse off the nonadherent cells, air dried, and dyed with the 0.01% acridine orange at 4 C for 20 min. Stained coverslips were gently rinsed 2 times with sterile PBS and observed with a CLSM (Nikon, Japan). Mature Biofilm Elimination Assay. We use fluorescent dyes to stain the living bacteria in the biofilm, and to determine the eliminating effect on the mature biofilm. 3 Firstly, 200 µl bacteria ( CFU/mL) growing in logarithmic phase was added to the holes of aseptic 96-well microtiter plate (5 wells per sample), and then cultivated the plate in 37 C incubator for 48 h to form mature biofilm. After the mature biofilm was formed, the bacteria solution from each well was removed, and the biofilm was washed with PBS buffer. Then, Au NCs was added into the holes with the final concentration of 0, 4, 8, 16, 32, 64, 128, and 256 µg/ml and further incubated at 37 C for 24 h, respectively. Next fluorescein diacetate (FDA) was used to stain the living bacteria in the biofilm. FDA solution of 100 µl was added to each hole and incubated at 37 C with dark environment for 1 h. The FDA solution placed in a clean hole was used as a blank sample. The fluorescence of each solution is recorded with microplate reader after an hour incubation (MK3, ThermoFisher). Membrane Integrity Test. The bacterial membrane integrity tests were determined using a commercial The LIVE/DEAD BacLight Bacterial Viability Kit (Invitrogen). The kit provides a two-color fluorescence assay of bacterial viability, when bacteria with intact cell membranes stain fluorescent green, whereas bacteria with damaged membranes stain fluorescent red. MDR E. coli or MRSA ( CFU/mL) were treated with Au NCs in LB at 37 C for 2 h, respectively. Then, the solutions were stained with SYTO 9 and PI and incubated for 30 min. The samples were observed using a CLSM (Nikon, Japan). Bacterial Morphological Characterization. The morphological changes of bacteria after S2

3 treatment with Au NCs were observed by using a SEM. The as-prepared bacterial solutions were centrifuged at 5000 rpm for 5 min, and then immobilized with 2.5% glutaraldehyde for 1.5 h, dehydrating with graded ethanol (30%, 50%, 70%, 90%, and 100%), and then imaged under a SEM. Drug Resistance Development. S. aureus ATCC29213 were used as a model strain for the drug resistance test. Drug resistance was induced by repeatedly treating bacteria with antimicrobial agents at sublethal doses. 4 Briefly, the MICs of Au NCs and vancomycin (control) against S. aureus ATCC29213 was determined through 30 days of growth. MIC was measured by using the method described above. S. aureus ATCC29213 pressured to sub-mic concentrations (1/3 of MIC at that particular generation) were allowed to regrow and reach a logarithmic growth phase before being used for MIC measurement of the subsequent generation. Development of drug resistance in S. aureus ATCC29213 was evaluated by recording changes in the MIC normalized to that of the first cell passage. ROS Assay. Bacteria cells (~ CFU/mL) were treated with Au NCs or Au NPs for 2 h at 37 C, respectively. Then total ROS were measured using ROS assay kit (Beyotime Institute of Biotechnology, China) under the guidance of the manufacturer s instructions. The bacterial samples were mixed with 10 µm 2,7 -dichlorofluorescein diacetate (DCF-DA) and incubated for 1 h at room temperature in the dark. The fluorescence intensity of solutions was registered with a Shimadzu RF-5301 PC fluorospectrophotometer using 488/525 nm excitation/emission wavelength. Oxidase-Like Property of Au Nanoantibiotics. For the oxidation of TMB, Au nanoantibiotics dispersions were added into 0.01 M TMB solution and then UV-Vis absorption spectra was recorded at different times using a Biomate 3S spectrophotometer (Thermo Fisher, USA). For the identification of the product of catalytic oxidation, a H 2 O 2 assay kit (Beyotime Institute of Biotechnology, China) was employed. Detailed experiments were conducted under the guidance of the manufacturer s instructions. Peroxidase-Like Property of Au Nanoantibiotics. For the oxidation of TMB by H 2 O 2, the Au nanoantibiotics dispersions were diluted with PBS as the substrate and then 10 µl of 100 mm TMB solution and 2 µl of H 2 O 2 (10 M) was added. Kinetic measurements were carried out by monitoring the absorbance at 653 nm on a Biomate 3S spectrophotometer (Thermo Fisher, USA). For the assay of the product, OH, TA (hydroxyterephthalic acid) was used as a probe, which could easily react with OH to form a highly fluorescent product (TAOH). In a typical procedure, TA was added into the reaction solution, and then the fluorescence spectra of the solutions were collected using a Shimadzu RF-5301PC fluoremeter (Japan). MTT Assay. L02 and AT II cells were purchased from KeyGen Biotech. Co. Ltd. (Nanjing, China) and were seeded in a sterile 96-well microtiter plates 8 h and subsequently incubated with different concentrations of DAMP-Au NCs in medium for 24 h at 37 C (5% CO 2 ), respectively. Then, 20 µl MTT (5 mg/ml) was added to wells and further incubated for 4 h. The cell supernatant was discarded and 150 µl of DMSO was added, and gentle shaking in the shaker for 10 min. The absorbance at 490 nm (A 490 ) of solutions was measured using a microplate reader (MK3, ThermoFisher). Cell viability (%) was expressed as A test / A control 100%, where A represents the absorbance at 490 nm. Hemolysis Assay. Hemolysis assays were performed according to previously reported method. 2 The red blood cells were obtained from human blood by centrifugation at 2000 rpm for 15 min, S3

4 gently washed for 3 times with normal saline, and the subsequent re-suspended by using normal saline to prepare erythocyte suspension with a hematocrit of 2%. Then, different concentrations of DAMP-Au NCs were incubated with isovolumetric 2% erythrocyte suspension at 37 C for 2 h. Triton X-100 (1%) and normal saline were employed as controls, respectively. After incubation, the supernatant was obtained by centrifugation at 3000 rpm for 5 min, and transferred to a 96-well plate. The absorbance of supernatant was measured at 450 nm (A 450 ). The hemolysis percentage was calculated as follows: Hemolysis (%) = (A test - A negative control ) / (A positive control - A negative control ) 100%, where A represents the mean of absorbance value at 450 nm. Histopathology. 4-week old BALB/c female mice were administered 200 µl DAMP-Au NCs (1 mg/ml) once daily. Mice treated with normal saline were tested in parallel as a negative control. After administration for 2 d, major organs (heart, liver, spleen, lung, and kidney) were fixed in 4% formalin solution, processed routinely into paraffin, and stained with H&E. The pathologies were examined using an optical microscope. S4

5 SUPPLEMENTARY DATA Figure S1. Fluorescent characterization of AuDAMP. (a) Excitation and emission spectra of Au NCs centered at 420 nm (blue line) and 620 nm (red line), respectively. Inset shows the photographs of AuDAMP under visible light (1) and 365 nm UV light (2). (b) Time resolved photoluminescence profiles of AuDAMP. Figure S2. The fluorescent stability of AuDAMP aqueous solution at room temperature. S5

6 Figure S3. The TEM image (a) and zeta potential (b) of DAMP stabilized Au NPs synthesized by reduction of NaBH 4. The average diameter is approximately 6 nm. The zeta potential is centered at mv. We stirred the mixture of DAMP (15 mm, 1 ml, dissolved in 50% ethanol solution) and HAuCl 4 (10 mm, 1 ml) for 10 min in the ice-water bath, added NaBH 4 (0.2 ml, 10 mm) with vigorous stirring. The solution turned brown immediately and then deep-red. We decreased the stirring speed and kept stirring the solution for 3 hours in the ice-water bath. The DAMP stabilized Au NPs was formed. Figure S4. Time-kill curves of AuDAMP against E. coli ATCC35218 (a), MDR E. coli (b), MDR A. baumannii (c), MDR K. pneumonia (d), MDR P. aeruginosa (e), S. aureus ATCC29213 (f), MRSA (g) and VRE (h) at different concentrations with different time. S6

7 Figure S5. MRSA biofilm formation was inhibited by AuDAMP. (a) Inhibition ratio of MRSA biofilm in the presence of different concentrations of AuDAMP. (b) CLSM image of biofilm formation by MRSA in the absence of (left) and the presence of (right) AuDAMP at 0.5 MIC concentration. The image shows the reconstructed 3-D biofilm images. Biofilm were stained with acridine orange, a widely used fluorescent biofilm biomass indicator. Scale bars = 25 µm. Figure S6. The effect of AuDAMP on a) planktonic bacteria and b) mature biofilm in MRSA model. S7

8 Figure S7. TEM and corresponding photo under 365 nm UV light (inset) images of AuDAMP and MDR E. coli (a) and MRSA (b) after co-incubation. As soon as the Au NCs come in contact with the bacteria, they are bounded on the bacterial cell surface, which in turn promotes internalization. Figure S8. Time-dependent absorbance spectra of the TMB in the presence of Au NPs. The characteristic absorption maximum of oxidized TMB locates at 653 nm. Au NPs hardly show any catalytic activity. S8

9 Figure S9. Time- and concentration-dependent absorbance changes at 653 nm of oxidized TMB in Au NCs reaction systems. Figure S10. Au NPs+H 2 O 2 +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: H 2 O 2 +TMB; Au NPs+TMB; Au NPs+H 2 O 2 +TMB. S9

10 Figure S11. The biocompatibility evaluations. (a) Cell viability of AT II and L02 cells treated with different concentrations of AuDAMP. (b) Hemolysis results and corresponding photo (inset) of different concentrations of AuDAMP. Normal saline (NaCl, 0.9%) and Triton X-100 solution (0.1%) were used for testing as negative control and positive control, respectively. (c) Histological evaluation of main organs (heart, liver, spleen, lung, and kidney) of mice treated with PBS buffer and AuDAMP. Figure S12. Photographs and the corresponding statistical histogram of bacterial colonies formed on the LB-agar plates derived from the lung tissue of mice injected with AuDAMP (5 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. S10

11 Table S1 Antibiotic susceptibility (MIC, µg/ml) of clinical A. baumannii, E. coli, K. pneumonia, P. aeruginosa, methicillin-resistant S. aureus (MRSA) and vancomycin-resistant E. faecium (VRE). Strain Antibiotics Class MIC Susceptibility A. baumannii E. coli K. pneumoniae Ciprofloxacin Quinolones 8 Resistant Levofloxacin Quinolones 8 Resistant Cefazolin Cephalosporins 64 Resistant Cefepime Cephalosporins 64 Resistant Cefoxitin Cephalosporins 64 Resistant Ceftazidime Cephalosporins 64 Resistant Ceftriaxone Cephalosporins 64 Resistant Macrodantin Nitrofurans 512 Resistant Amikacin Aminoglycosides 2 Sensitive Gentamicin Aminoglycosides 1 Sensitive Tobramycin Aminoglycosides 1 Sensitive Aztreonam Monobactams 64 Resistant Colistin Polymyxins 4 Resistant Ampicillin Penicillins 32 Resistant Imipenem Carbapenems 16 Resistant Tigecycline Tetracyclines 2 Sensitive Amoxicillin/ Clavulanic acid Penicillin combinations 32 Resistant Ampicillin/ Sulbactam Penicillin combinations 16/32 Resistant Piperacillin/ Tazobactam Penicillin combinations 128/16 Resistant Cefazolin Cephalosporins 16 Resistant Cefuroxime Cephalosporins 32 Resistant Cefepime Cephalosporins 32 Resistant Cefoxitin Cephalosporins 16 Resistant Ceftazidime Cephalosporins 32 Resistant Ceftriaxone Cephalosporins 64 Resistant Amikacin Aminoglycosides 1 Sensitive Gentamicin Aminoglycosides 1 Sensitive Colistin Polymyxins 2 Sensitive Ampicillin Penicillins 32 Resistant Chloramphenicol Chloramphenicols 32 Resistant Minocycline Tetracyclines 4 Sensitive Ciprofloxacin Quinolones 4 Resistant Levofloxacin Quinolones 32 Resistant Imipenem Carbapenems 1 Sensitive Meropenem Carbapenems 1 Sensitive Trimethoprim/sulfamethoxazole Sulfonamides 8/128 Resistant Cefoperazone/ Sulbactam Cephalosporin combinations 16/8 Resistant Piperacillin/ Tazobactam Penicillin combinations 4/4 Sensitive Ticarcillin / Clavulanic acid Penicillin combinations 128/2 Resistant Ampicillin / Sulbactam Penicillin combinations 32/16 Resistant S11

12 P. aeruginosa MRSA Amikacin Aminoglycosides 1 Sensitive Gentamicin Aminoglycosides 1 Sensitive Amikacin Aminoglycosides 2 Sensitive Tobramycin Aminoglycosides 1 Sensitive Aztreonam Monobactams 64 Resistant Ciprofloxacin Quinolones 32 Resistant Levofloxacin Quinolones 16 Resistant Moxifloxacin Quinolones 8 Resistant Cefepime Cephalosporins 64 Resistant Cefoxitin Cephalosporins 16 Resistant Ceftazidime Cephalosporins 32 Resistant Ceftriaxone Cephalosporins 32 Resistant Colistin Polymyxins 1 Sensitive Ampicillin Penicillins 32 Resistant Tetracycline Tetracyclines 16 Resistant Ertapenem Carbapenems 0.5 Sensitive Imipenem Carbapenems 1 Sensitive Meropenem Carbapenems 1 Sensitive Trimethoprim/sulfamethoxazole Sulfonamides 16 Resistant Ampicillin / Sulbactam Penicillin combinations 8/16 Resistant Piperacillin/ Tazobactam Penicillin combinations 4 Sensitive Amikacin Aminoglycosides 16 Intermediate resistance Gentamicin Aminoglycosides 4 Sensitive Tobramycin Aminoglycosides 1 Sensitive Ciprofloxacin Quinolones 8 Resistant Levofloxacin Quinolones 16 Resistant Colistin Polymyxins 0.5 Sensitive Cefazolin Cephalosporins 64 Resistant Cefepime Cephalosporins 8 Resistant Ceftazidime Cephalosporins 32 Resistant Ceftriaxone Cephalosporins 32 Resistant Aztreonam Monobactams 32 Resistant Imipenem Carbapenems 16 Resistant Ampicillin Penicillins 16 Resistant Trimethoprim/sulfamethoxazole Sulfonamides 4/32 Resistant Ampicillin / Sulbactam Penicillin combinations 8/2 Sensitive Piperacillin/ Tazobactam Penicillin combinations 4/2 Sensitive Erythromycin Macrolides 8 Resistant Clindamycin Lincosamides 16 Resistant Ciprofloxacin Quinolones 0.5 Sensitive Levofloxacin Quinolones 16 Resistant Moxifloxacin Quinolones 0.5 Sensitive Linezolid Oxazolidinones 1 Sensitive S12

13 VRE Cefepime Cephalosporins 64 Resistant Cefoxitin Cephalosporins 32 Resistant Amikacin Aminoglycosides 64 Resistant Gentamicin Aminoglycosides 16 Resistant Vancomycin Glycopeptides 2 Sensitive Methicillin Penicillins 64 Resistant Oxacillin Penicillins 64 Resistant Penicillin G Penicillins 4 Resistant Rifampicin Rifamycins 0.5 Sensitive Trimethoprim/sulfamethoxazole Sulfonamides 8 Sensitive Tetracycline Tetracyclines 16 Resistant Clindamycin Lincosamides 2 Sensitive Amikacin Aminoglycosides 16 Resistant Gentamicin Aminoglycosides 32 Resistant Tobramycin Aminoglycosides 32 Resistant Tetracycline Tetracyclines 16 Resistant Erythromycin Macrolides 1 Sensitive Ciprofloxacin Quinolones 16 Resistant Levofloxacin Quinolones 32 Resistant Moxifloxacin Quinolones 16 Resistant Linezolid Oxazolidinones 2 Sensitive Cefoxitin Cephalosporins 8 Resistant Teicoplanin Glycopeptides 16 Resistant Vancomycin Glycopeptides 32 Resistant Ampicillin Penicillins 16 Resistant Oxacillin Penicillins 32 Resistant Penicillin G Penicillins 4 Resistant Rifampicin Rifamycins 2 Sensitive Trimethoprim/sulfamethoxazole Sulfonamides 8 Sensitive Table S2 MIC based on surface DAMP ligand concentrations (µg/ml). AuDAMP NCs AuDAMP NPs E. coli ATCC MDR E. coli MDR A. baumannii MDR K. pneumoniae MDR P. aeruginosa S. aureus ATCC MRSA VRE Note: The EDS 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. By conversion, the corresponding mass ratio of Au-to-DAMP is 1.4:1 and 1:1, respectively. This ratio is used to normalize the ligand concentrations during comparison of the MIC. S13

14 References (1) Li, S., Wang, Z., Wei, Y., Wu, C., Gao, S., Jiang, H., Zhao, X., Yan, H., Wang, X. (2013) Antimicrobial Activity of A Ferrocene-Substituted Carborane Derivative Targeting Multidrug-Resistant Infection. Biomaterials 34, (2) Zheng, Y., Liu, W., Chen, Y., Jiang, H., Yan, H., Kosenko, I., Chekulaeva, L., Sivaev, I., Bregadze, V., Wang, X. (2017) A Highly Potent Antibacterial Agent Targeting Methicillin-Resistant Staphylococcus aureus Based On Cobalt Bis(1,2-dicarbollide) Alkoxy Derivative, Organometallics 36, (3) Xie, Y., Liu, Y., Yang, J., Liu, Y., Hu, F., Zhu, K., Jiang, X. (2018) Gold Nanoclusters for Targeting Methicillin-Resistant Staphylococcus aureus In Vivo. Angew. Chem. Int. Ed. 57, (4) Li, X., Robinson, S. M., Gupta, A., Saha, K., Jiang, Z., Moyano, D. F., Sahar, A., Riley, M. A., Rotello, V. M. (2014) Functional Gold Nanoparticles as Potent Antimicrobial Agents Against Multi-Drug-Resistant Bacteria. ACS Nano 8, S14