Indole Derivatives-Capped Gold Nanoparticles as Effective

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Biological and Medical Applications of Materials and Interfaces

Indole Derivatives-Capped Gold Nanoparticles as Effective Bactericide in vivo Xiaohui Zhao, Yuexiao Jia, Juanjuan Li, Ruihua Dong, Jiangjiang Zhang, Chuanxin Ma, Hui Wang, Yu-kui Rui, and Xingyu Jiang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b11980 • Publication Date (Web): 07 Aug 2018 Downloaded from http://pubs.acs.org on August 8, 2018

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ACS Applied Materials & Interfaces

Indole Derivatives-Capped Gold Nanoparticles as Effective Bactericide in vivo Xiaohui Zhao †,§,‡, Yuexiao Jia §,#,‡, Juanjuan Li §, Ruihua Dong §, Jiangjiang Zhang §,# , Chuanxin Ma ¦, Hui Wang ǂ, Yukui Rui †,*, Xingyu Jiang §,#,*

†

Beijing Key Laboratory of Farmland Soil Pollution Prevention and Remediation,

College of Resources and Environmental Sciences, China Agricultural University, Beijing100193, China §

Beijing Engineering Research Center for BioNanotechnology and CAS Key Laboratory

for Biomedical Effects of Nanomaterials and Nanosafety, CAS Center for Excellence in Nanoscience, National Center for NanoScience and Technology, No. 11 Zhongguancun Beiyitiao, Beijing 100190, P. R. China #

University of Chinese Academy of Sciences, 19 A Yuquan Road, Shijingshan District,

Beijing, 100049, P. R. China ¦

Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station,

New Haven, CT 06504, United States ǂ

Department of Clinical Laboratory, Peking University People's Hospital, Beijing 100044,

China

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ABSTRACT: We synthesize indole derivatives-capped gold nanoparticles (Au_IDs) via a straightforward route to fight multi-drug resistant (MDR) bacteria. When gold nanoparticles are modified with two indole derivatives, tryptophan and 5-aminoindole, they exhibit excellent antibacterial activities against both laboratory antibiotic-sensitive and MDR bacteria. Au_IDs possess remarkable bactericidal activities against MDR bacteria killing 99.9% of MDR Escherichia coli and polymyxins-resistant Klebsiella pneumonia after 0.5 h incubation, which are superior to clinical antibiotics including polymyxin B and cefotaxime. By evaluating the potential of Au_IDs in wound cure, Au_IDs show outstanding capability in MDR bacteria wound infection. Our findings provide new candidates for the development of bactericides and the fabrication of wound dressings for treating MDR infection. KEYWORDS: indole derivatives; gold nanoparticles; MDR infection; bactericide; wound healing

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1. INTRODUCTION Infectious disease caused by Gram-negative bacteria is one of the greatest global health problems, tormenting millions of people every year. Many types of infections can be caused by Gram-negative bacteria. For example, Escherichia coli (E. coli) can induce infections of gastrointestinal and urinary tract;1 Klebsiella pneumoniae (K. p) can cause liver abscess.2 Antibiotics have been widely used by humans to treat infections since their first discovery in 1920s.1 Unfortunately, the extensive usage and abuse of antibiotics rapidly resulted in drug resistance which brought new social panic.3 Carbapenems have been the main treatment for multi-drug resistant (MDR) Gram-negative bacteria over the past decade. However, some studies have reported that the resistance of drugs makes carbapenems lose efficacy. Polymyxins, which are the last line of defense for MDR bacteria, are also extremely limited in the cure of carbapenems-resistant bacterial infections.4,5 Research for new antibiotics with better antibacterial properties has been going on constantly. However, far fewer new antibiotics have been discovered and approved in the past decade than at the peak in the 1980s.6 Due to the growing clinical and market demand, the development of new antimicrobial agents is urgent. Nanoscience and nanotechnology hold great promise for antibacterial applications, including medical devices, safe cosmetics, burn dressings, water treatment, food preservation, and other products.7 Scientists have developed various nanomaterials, including Ag, CuO, ZnO and TiO2 nanoparticles (NPs), for antibacterial applications.8-12

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Although these NPs can fight against bacteria, they have some drawbacks like high toxicity or unsatisfactory antibacterial effects, which hinder their application for antibiotic drugs.13,14 Gold nanoparticles (Au NPs) have been reported in various bio-applications like antibacterial application,15-17 cancer therapeutics,18-20 antithrombotic treatment21 and biochemical analysis.22-25 Au NPs are promising antibacterial agents, due to their nontoxicity, polyvalent effects and easy functionalization.26-28 In our group, we have developed kinds of Au NPs to fight MDR bacteria. We use none-antibiotic small molecules, like pharmaceutical intermediates, N-heterocyclic molecules, and amines to modify Au NPs for antibacterial applications in vitro and in vivo.29-32 The Au NPs have excellent antibacterial activities against not only laboratorial strains but also clinical MDR isolates, and they are low toxicity to human cells. In this work, we try to search some other small molecules to modify Au NPs for curing MDR infections. Indole is an important class of compounds widely used in many fields including food additives, fragrances and pesticide intermediates. The indole moiety is commonly involved in clinical drugs such as naratriptan, zolmitriptan, rizatriptan, eletriptan, and almoteiptan.33 Moreover, indole is also used for designing new drugs for antifungal agents, antitumor drugs and anti-HIV-1 agents.34-36 Some studies have obtained antibacterial compounds from indole derivatives by chemical reactions.37,38 However, the synthesis of these compounds involves a variety of complex reactions. An indole derivative, tryptophan, has been used as reducing/stabilizing agent to prepare Au NPs.39,40

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But the antibacterial applications of the Au NPs have not been explored. In this study, we try to use indole derivatives (with no antibacterial activity) to modify Au NPs for antibacterial applications. In this work, we synthesize indole derivatives-capped gold nanoparticles (AI_IDs) based on a simple one-pot method using indole derivatives as reductants and ligands at room temperature. Our method possesses several advantages like simple operation and mild reaction conditions. When Au NPs are modified with tryptophan (W) and 5-amindole (5-AI), they (Au_W and Au_AI) exhibit outstanding antibacterial performance. The minimum inhibitory concentration (MIC) is 2 µg/mL against most tested bacteria, even clinical MDR bacteria. Au_W and Au_AI are extremely effective in killing MDR bacteria even at high bacterial concentrations. After 0.5 h incubation, they can kill 99.9% of MDR Escherichia coli (MDR E. coli) and polymyxins-resistant Klebsiella pneumoniae (PR K. p). More than 99.99% of PR K. p will die after 4 h incubation with Au_AI even with a high initial bacterial concentration (bacterial concentration: 5×108 CFU/mL). Bactericidal activities are necessary for antibiotics to fight serious bacterial infections, because they not only can avoid serious bacterial infections but also can reduce the occurrence of drug resistance.41 Our work is the first example that comprehensively evaluates the bactericidal performance of Au NPs. Besides, we fabricate Au_AI fibers as wound dressing by electrospinning for the treatment of MDR bacterial wound infections. Our fibers can accelerate wound healing, which is

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better than commercial gauze (Figure 1). Thus, we believe that our findings provide a new kind of nanomaterial as potential bactericidal agent.

2. EXPERIMENTAL SECTION 2.1 Materials. HAuCl4·4H2O was from Beijing Puyihua Science and Technology Co., Ltd., China. Indole (ID), tryptophan (W), 4-aminoindole (4-AI), 5-aminoindole (5-AI), 6-aminoindole (6-AI), 7-aminoindole (7-AI), 5-indolylboronic (5-BI), 5-chloroindole (5-CLI), indole-3-acetic acid (IAA), 4-hydroxyindole (4-HI), 5-cyanoindole (5-CNI), and 3-sulfanylindole (3-SI) were from Sigma. Several standard strains we used including Escherichia coli (E. coli), Klebsiella pneumoniae (K. p), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. a) were obtained from Beijing LABLEAD Co. Ltd., China. The clinical isolates include polymyxins-resistant Escherichia coli (PR E. coli), multidrug-resistant Escherichia coli (MDR E. coli), polymyxins-resistant Klebsiella pneumoniae (PR K. p), multidrug-resistant Acinetobacter baumannii (MDR A. b) and multidrug-resistant Klebsiella pneumoniae (MDR K. p) were from local hospitals in China. Human umbilical vein endothelial cells (HUVECs) were purchased from Guangzhou Xiangbo Biotechnology Co. Ltd., China. Polymyxin B sulfate (Code: P815744-1 g, Lot#: C10052738) was from Macklin. Cefotaxime sodium salt (Code: C804340-250 mg, Lot#: C10009602) was from Macklin. SYTO 9/PI and cell counting kits (CCK-8) were from Sigma. Sprague-Dawley (SD) male rats (4 weeks, 200 g) were obtained from Beijing HFK Bioscience Co. Ltd., China. 2.2 Synthesis of Indole Derivatives-capped Gold NPs. Indole derivatives-capped gold nanoparticles (Au_IDs) were synthesized by reducing HAuCl4 with indole or its derivatives (IDs) as both reductants and ligands.40,42 The indole derivatives (IDs) we used 6


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here include ID, W, 4-AI, 5-AI, 6-AI, 7-AI, 5-BI, 5-CLI, IAA, 4-HI, 5-CNI, and 3-SI. 1 mL of HAuCl4·4H2O (41 mg/mL) and 1.25 mL of Tween 80 solution (1 mg/mL) were mixed together. Indole solution (5 mmol/L, 10 mL) was added dropwise until the color of the solution did not change and the solution was stirring for another 15 minutes. The solution was transferred into a dialysis bag (Molecular weight cutoff 14 kDa, Millipore) and placed in a beaker containing pure water for 48 hours. After centrifuging the solution at 5000 rpm for 10 min, the supernatant (Au_ID) was collected for the following experiment. Au_W, Au_4-AI, Au_5-AI, Au_6-AI, Au_7-AI, Au_5-BI, Au_5-CLI, Au_IAA, Au_5-CNI, Au_4-HI and Au_3-SI were synthesized through the same method. 2.3 Characterization of Au_IDs. Transmission electron microscope (TEM, Tecnai G2 20, FEI) was used to characterize the morphologies of Au_IDs. Inductively coupled plasma optical emission spectrometer (ICP-OES, iCAP 6300, Thermo Scientific) was used to quantify the Au concentrations in Au_IDs. The Au concentrations of Au_W and Au_AI were 257 µg/mL (1.30 mM) and 260 µg/mL (1.32 mM), and the nanoparticles concentrations of Au_W and Au_AI were 0.14 µM and 0.19 µM, respectively (Table S2). Dynamic light scattering (DLS, Zetasizer Nano ZS, Malvern) provided zeta potential (surface charge) and hydrodynamic size (radii in an aqueous solution) of Au_IDs. Fourier transform infrared spectroscopy (FTIR, Spectrum One, Perkin Elmer) and X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Fisher Scientific) helped to further characterize the chemical/elemental composition of Au_IDs. Nuclear magnetic resonance spectrometer (NMR, AVANCE III HD 400, American Bruker) was used to measure 1H NMR spectra of IDs and Au_IDs (dissolved in DMSO-d6). The data of 1H 7



NMR spectra were referenced the Spectral Database for Organic Compounds (SDBS) on http://sdbs.db.aist.go.jp (National Institute of Advanced Industrial Science and Technology, date of access). 2.4 Antibacterial Experiment. The microbroth dilution method can determine the MIC and the minimal bactericidal concentration (MBC) of Au_IDs. 100 µL of nutrient broth was added to each well in 96-well plate and 100 µL of Au IDs was added into the first column of the plate. Stepwise diluted Au IDs from the first column to penultimate column served as the experimental group, while the last column served as the blank control. 10 µL of bacteria (concentration: ~105-106 CFU/mL) was added into each well of the plate. The plate was incubated at 37 oC for 24 h and the MICs were recorded as the minimal concentration of the drug that cannot cause the turbidity of the bacterial suspensions. The bacterial suspensions in the wells which were clear were taken onto agar plates. The plates were cultured at 37 oC and observed after 24 h. The MBCs were recorded as the minimal concentration of the drug that can eliminate bacteria by 3 orders of magnitude. To study the synergistic effect of Au NPs and IDs, the antibacterial activities of the mixture of Au NPs and IDs was tested. The Au NPs was synthesized by the same method using sodium borohydride instead of IDs as a reducing agent. After mixing 50 µL of Au NPs and 50 µL of IDs, the mixture (the final concentration of Au NPs is 174.5 µg/mL, the final concentrations of W and AI are 408.5 µg/mL and 264.3 µg/mL) was assayed for MIC. 2.5 Bactericidal Assay. 2.5.1 Concentration-dependent bactericidal activity of Au_IDs. The overnight cultured MDR E. coli were washed by sterile phosphate buffered saline 8


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(PBS) twice and re-suspended to PBS with OD600 nm=0.5 (~108-109 CFU/mL). The bacteria were diluted 1000 times (final bacterial concentration: 2×105 CFU/mL) and treated with PBS (control), Au_W (16, 8, 4, 2, 1 µg/mL), Au_AI (16, 8, 4, 2, 1 µg/mL) in culture tubes at 37 oC. After 12 h, the bacterial suspensions were diluted for 10 times and added 100 µL of the diluted suspensions onto the nutrient broth agar plates and incubated at 37 oC. After 24 h, CFU/mL was calculated by counting colonies. 2.5.2 Time-dependent bactericidal activity of Au_IDs. The time-dependent bactericidal activities of Au_W (8 µg/mL, 0.004 µM), Au_AI (8 µg/mL, 0.006 µM), polymyxin B (32 µg/mL, 27 µM), cefotaxime (32 µg/mL, 70 µM) and PBS (control) were tested against MDR bacteria (MDR E. coli and PR K. p) at different bacterial concentration (low bacterial concentration: 2×105 CFU/mL; high bacterial concentration: 5×108 CFU/mL). At selected time points (low: 0, 0.5, 4, 8, 12 h; high: 0, 2, 4, 8, 12, 24 h), the bacterial suspensions were taken onto the agar plates. After incubating the plates at 37 oC for 24 h, the results were recorded. 2.6 Confocal Imaging of Bacteria. 1 mL of bacteria (MDR E. coli and PR K. p, respectively, 5.0 × 108 CFU/mL) were cultured with and without Au_AI and Au_W (8 µg/mL) at 37 °C for 4 h. After centrifuging the bacterial suspensions (8000 rpm, 3 min), the bacteria were collected. The bacteria were washed with PBS twice, and stained with SYTO 9 and propidium iodide (PI) solution (37 °C, 30 min). The bacteria were washed with PBS and observed by a confocal microscope (Zeiss 710, Zeiss). 2.7. Bacteria Preparation for SEM and TEM. E. coli was incubated with PBS (control), Au_AI and Au_W (8 µg/mL, at 37 °C for 4 h) for scanning electron microscope (SEM, Hitachi-SU8220, Hitachi) and TEM characterizations. The bacteria were fixed 9



with 2.5% glutaraldehyde for 12 h and dehydrated in different concentration of ethanol (30, 50, 70, 80, 90 and 100% v/v, in water) according to literature.29 The morphologies of bacteria were observed by SEM. The Au_IDs treated bacterial samples were placed on cupper grid and stained by uranyl acetate for TEM imaging. 2.8 Cytotoxicity of Au_IDs. The HUVECs were cultured in DMEM with 1% penicillin/streptomycin and 10% fetal bovine serum. HUVECs were incubated in the 96-well plate (100 µL, 1×105 cells per milliliter) at 37 °C for 12 h to allow their attachment on the plate. Different concentrations (0, 2, 4, 8, 16, 32 and 64 µg/mL, dissolved in DMEM) of Au_IDs were added into the plates. The plates were incubated at 37 °C for 24 h. The HUVECs in the plates were washed with PBS and stained with 10 % (v/v) CCK-8. After 2 h, the optical density at 450 nm (OD450 nm value) of each well was measured using a microplate reader (Tecan infinite M200). 2.9 Preparation of Au_AI Electrospun fibers. Au_AI nanoparticles were synthesized in methanol. After the completion of synthesis, the solvent of Au_AI solution was eliminated at 40 °C in vacuum. The residue and poly (lactic-co-glycolic acid) (PLGA) were added into dichloromethane (4 g) and the mixture was stirred for 4 h to make a solution (the final concentration of Au_AI was 10.75 µg/mg, and the final concentration of PLGA was 20 wt%). The fibers were fabricated by electrospinning the solution at 15.0 kV and collected at the distance of 12 cm. PLGA fibers were prepared using the same method expect the addition of Au_AI. 2.10 Rat Wound Model and Histological Analysis. A rat wound model was built to evaluate the antibacterial activity of Au_IDs in vivo. Three round wounds with the same size (d = 1.5 cm) were cut on the back of each rat. The wounds were infected by MDR E. 10


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coli (1×108 CFU/mL, 100 µL) for 30 min. Then, the three wounds were covered with gauze, PLGA, and Au_AI fibers. On day 3, 6 and 12, each wound was photographed and their sizes were measured. On day 6 and 12, the wound tissues were cut off and dipped in fixative (4% paraformaldehyde). The wound tissues were handed to the company for slicing and staining (Beijing Youboao Biotechnology Co. LTD., China).

3. RESULTS AND DISCUSSION 3.1 Synthesis and Antibacterial Activities of Au_IDs. We synthesize a series of Au_IDs using IDs as both stabilizing and reducing agents and evaluate the antimicrobial activities of Au_IDs against bacteria (Gram-negative bacteria: E. coli, Gram-positive bacteria: S. aureus) by dilution method (Table 1, Table S1). The Au_IDs (Au_ID, Au_W, Au_4-AI, Au_5-AI, Au_6-AI, Au_7-AI, Au_5-BI, Au_5-CLI, Au_IAA, Au_5-CNI, Au_4-HI and Au_3-SI) are active against Gram-negative bacteria, and the MIC varies from 4 to 30 µg/mL. In contrast, they have no antibacterial effect on Gram-positive bacteria. The IDs (ID, W, 4-AI, 5-AI, 6-AI, 7-AI, 5-BI, 5-CLI, IAA, 4-HI, 5-CNI, and 3-SI) by themselves have no antibacterial activities on neither Gram-positive nor Gram-negative bacteria. Au NPs without any surface modification have no antibacterial activity (Table S4). This means that the modified IDs on the gold nanoparticles gives them antibacterial properties. For Au NPs, surface modification is important for their functions. In our previous studies, non-antibiotic molecules, like pharmaceutical intermediates, N-heterocyclic molecules, and amines, are modified on Au NPs to yield antibacterial activities.29-32 In this work, IDs-modifications can also give antibacterial 11



properties to Au NPs. Moreover, adjustment of the surface modification can regulate the antibacterial properties of the Au NPs. For example, 4,6-diamino-2-pyrimidinethiol modified gold nanoparticles (Au_DAPT) have antibacterial effect only on Gram-negative bacteria. After adjusting the surface modification of Au NPs (add another ligand, 1,1-dimethylbiguanide, DMB), the gold nanoparticles (Au_DAPT/DMB) can fight both Gram-negative bacteria and Gram-positive bacteria.32,43 Unlike the previous ligand, the modification of IDs gives the Au_IDs excellent antibacterial and bactericidal performance. When W and 5-AI modify Au NPs, the Au NPs (Au_W and Au-AI) show the best antibacterial activities against E. coli and the MIC is 4 µg/mL. Thus, we choose Au_W and Au_AI for further studies. 3.2 Characterization of Au_IDs. To study the properties of Au_IDs, we characterize Au_W and Au_AI, which are the two most efficient antibacterial nanoparticles. They are water soluble, and their colors are purple and brown (inset in Figure 2A). The UV-Vis spectrum indicates that Au_W has an obvious absorption peak at 522 nm, which is due to the surface plasmon resonance (SPR) of Au NPs (Figure 2A). TEM images show that Au_W and Au_AI are uniformly dispersed nanoparticles with the sizes of 6.02±0.22 nm and 5.55±0.15 nm, respectively (Figure 2B). The hydrodynamic sizes of Au_W and Au_AI are 10.55±0.21 nm and 6.32±0.34 nm, and the NPs are negatively charged with zeta potential of -16.3±2.93 mV and -27.6±1.08 mV (Table S2). We can observe the characteristic peak of W and AI (1367-740 cm-1: the pyrrole and benzene rings; 3304 cm-1:

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the N-H in the indole ring) in the FTIR spectra of Au_W and Au_AI (Figure 2C).40 The Au and N peaks appear simultaneously in the XPS spectra (Figure 2D). These results indicate that the IDs are successfully modified onto the nanoparticles.30 The high-resolution Au 4f XPS spectra show that Au_W and Au_AI have both Au (0) and Au (I), and the ratio of Au (I): Au (0) in Au_AI is higher than that in Au_W. It is reported that the high ratio of Au (I): Au (0) in the nanoparticles will cause the absence of the SPR of the Au NPs, leading to the disappearance of UV-Vis absorption peak of them (Figure S1).44 Some Au (I) complexes have catalytic and biological potential due to their structural features and are used in some applications of antibacterial, antitumor and rheumatoid arthritis.45-47 Thus, we believe that the Au (I) can contribute to the antibacterial activity of Au_IDs. To further study the ligands on the surface of Au_IDs, 1H NMR spectra are measured. In pure W and AI, the multiple peaks representing aromatic protons of IDs appear in the 6-8 ppm range (Figure 2E). These peaks in the spectra of Au_W and Au_AI are significantly reduced, which indicate that the indole groups in the ligand molecules on the surface of the nanoparticles have changed.39 It is reported that the reduction of the number of aromatic protons on the indole ring is due to the oxidative polymerization of ligand molecules. Indole molecules are commonly used in chemical synthesis, and the indole groups were oxidized and polymerized when the oxidant was present, such as FeCl3 and CuCl2.48 In our work, IDs may be oxidized by HAuCl4 and polymerized on the surface of Au nanoparticles.

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3.3 Antibacterial Activities. The MICs of Au_W and Au_AI against several representative species of Gram-negative bacteria including clinical MDR strains are evaluated. The Au_IDs have antibacterial activities against all of the tested Gram-negative bacteria (Table 2). The MIC can be as low as 2 µg/mL against MDR E. coli, PR E. coli and K. p. The antibacterial activities of Au_IDs are also evaluated by measuring the optical density at 600 nm (OD600 nm) and observing the turbidity of the bacterial suspensions (Figure S2, S3). Au_W and Au_AI can suppress the increase of OD600 nm and turbidity of both antibiotic-sensitive bacteria and MDR strains which indicate the inhibition of bacterial proliferation. These clinical MDR strains have resistance to many antibiotics. For example, MDR E. coli are resistant to most antibiotics, including oxacillin, penicillin, cefotaxime and gentamicin, and are sensitive to only two antibiotics, polymyxins and carbapenem (Table S3). For PR K. p, polymyxins and carbapenem are ineffective, and only cefotaxime has effect on PR K. p. Thus, Au_W and Au_AI can be potent antibiotic reagents for the treatment of MDR infection. To determine if the Au_IDs is bactericidal, the MBC against bacteria is also tested. MBC is the minimal concentration required to eliminate bacteria by 3 orders of magnitude. Au_W has MBCs of 4 µg/mL against K. p and MDR E. coli, and 8 µg/mL against E. coli and PR K. p. Au_AI shows MBCs of 4 µg/mL against K. p, and 8 µg/mL against E. coli, PR E. coli, PR K. p, MDR E. coli and MDR A. b. Antibiotics with MBC/MIC no more than 4 are defined as bactericidal agents.31,49 The MBC/MIC in our

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study are no more than 4, which means that Au_W and Au_AI belong to bactericides against most tested Gram-negative strains. To validate the synergistic effect between Au NPs and IDs against bacteria, we measured the MIC and MBC of W, AI and unmodified Au NPs toward E. coli (Table S4). Au NPs and the IDs (W and AI) have no antibacterial activity themselves (Au NPs: MIC>349 µg/mL, W: MIC>5000 µg/mL, AI: MIC>5000 µg/mL, which are far more than the doses used in clinical antibiotics). The mixture of W and AI with Au NPs (Au NPs+W and Au NPs+AI) have no antibacterial activity, either. However, W and AI modified gold nanoparticles (Au_W and Au_AI) exhibit excellent antibacterial effects on E. coli. The MBCs indicate that W, AI, Au NPs, Au NPs+W and Au NPs+AI have no bactericidal activities, while Au_W and Au_AI have bactericidal effects on E. coli. In addition, the OD600 nm values also show that W, AI, Au NPs, Au NPs+W and Au NPs+AI do not inhibit the growth of bacteria, while Au_W and Au_AI can suppress the growth of bacteria (Figure S4). These results show that the antibacterial activity can only be produced by the modification of IDs onto Au NPs. 3.4 Bactericidal Activities. We evaluate the bactericidal properties of Au_IDs. The MDR E. coli is used as a microbial model to investigate the concentration-dependent killing of MDR bacteria (Figure S5, the initial bacterial concentration: 2×105 CFU/mL). At 8 µg/mL, Au_W and Au_AI can eliminate 99.9% bacteria after 12 h. Even at 1 µg/mL,

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Au_W and Au_AI can kill more than 90% bacteria. The results show that Au_W and Au_AI possess excellent bactericidal effect on MDR bacteria. To further study the bactericidal properties of Au_IDs, the time-dependent killing of two MDR strains (MDR E. coli and PR K. p) by Au_W (8 µg/mL), Au_AI (8 µg/mL) and two antibiotics (polymyxin B: 32 µg/mL, cefotaxime: 32 µg/mL) are compared. Au_W and Au_AI can kill 99.9% of MDR E. coli and PR K. p after 0.5 h incubation at low bacterial concentration (bacterial concentration: 2×105 CFU/mL) (Figure 3A, S6 and S7). However, antibiotics have no effective bactericidal effect on the two MDR bacteria. For example, polymyxin B and cefotaxime can only kill 9.99% and 0.1% of MDR E. coli after 4 h incubation, respectively. Polymyxin B has no effect on PR K. p. When a large number of bacteria infect the wound, it can delay wound healing and even cause the sepsis.50,51 We wonder if Au_IDs could effectively kill MDR bacteria at high concentrations of bacteria. For high bacterial concentration (bacterial concentration: 5×108 CFU/mL), Au_AI can kill 99.99% of MDR E. coli after 8 h incubation, and Au_AI can kill 99.99% of PR K. p after 4 h (Figure 3B). Au_W can kill 99.96% of PR K. p after 4 h incubation. Polymyxin B and cefotaxime have almost no bactericidal effect on PR K. p. Thus, Au_W and Au_AI are superior to antibiotics in killing bacteria at both low and high bacterial concentrations. These results indicate the Au_IDs could become potential bactericides which can kill bacteria rapidly. Au_IDs possess excellent bactericidal performance due to the modification of IDs. Our synthesized gold nanoparticles have

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better bactericidal efficacy than previous gold nanoparticles.29-32 Bactericidal agents are beneficial in clinical treatment of serious bacterial infections. When a serious bacterial infection occurs, especially if the wound is infected with a large number of bacteria, it is necessary to have a fast and effective bactericide that can kill the bacteria in a short time and prevent the bacteria from proliferation. Our synthesized nanoparticles can quickly kill bacteria, especially MDR bacteria, and its bactericidal effect is far better than antibiotics. Thus, Au_IDs have potential usefulness for clinical treatment, especially the treatment of serious bacterial infections. The bactericidal activities of Au_IDs are further confirmed by staining the bacteria with SYTO 9/PI based on the BacLight method.31 SYTO 9 can stain DNA or RNA by crossing the membrane of viable cells (green fluorescence). PI cannot cross the intact cell membrane and can only stain DNA or RNA when the cell membrane is damaged (red fluorescence). The suspensions of MDR E. coli and PR K. p are treated with Au_W and Au_AI (the final concentration, 8 µg/mL) at 37 oC. After 4 h, the samples are stained with SYTO 9/PI and imaged with confocal microscope. The appearance of large amounts of green fluorescence in untreated bacteria means that the cell membranes of most bacteria are intact (Figure 3C). Fluorescence images show that red fluorescence appears in large quantities of the bacteria treated with Au_W and Au_AI, which implies increased cell membrane permeability of bacteria. The results show that the presence of Au IDs changes the membrane permeability of bacteria. The phenomena may provide direction for

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understanding the antibacterial mechanism of nanoparticles. 3.5 Antibacterial Mechanisms of Au_IDs. Based on the above findings, we speculate that the death of bacteria treated with Au_IDs is due to the destruction of the cell membrane. To confirm the speculation, the morphology of E. coli treated with Au_W and Au_AI are observed (Figure 4). SEM images show that the cell membrane of the untreated E. coli is intact. After incubated E. coli with Au_W and Au_AI for 4 h, cell lysis of E. coli is observed. Au_IDs mainly affect the morphology of the bacteria and cause a collapse at the middle of the bacteria. TEM images show that the cell membrane of the untreated bacteria is integral. However, after the treatment of Au_W and Au_AI, the cell membranes of the bacteria are completely broken. Hence, Au_W and Au_AI can induce disruption to cell membrane, which could be the main antibacterial mechanisms of Au_W and Au_AI. 3.6 In vivo Application of Au_IDs. After evaluating the cytotoxity of Au_IDs (Figure S8), the wound-healing capability with MDR bacterial infection of Au _IDs are tested. Au_AI fibers are prepared by co-electrospinning of PLGA and Au_AI. Au_AI is chosen because Au_AI have the best bactericidal ability. PLGA has good biocompatibility, biodegradability and film-forming ability, which might be conducive to wound treatment.52,53 The SEM images of fibers show that these fibers are uniformly distributed, uniform in size, and the diameter is about 1 µm (Figure S9). To investigate the efficiency of Au_AI fibers, a model of wound infection (three dorsal wounds on each rat) by MDR

18


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E. coli is built. Gauze, PLGA fibers, and Au_AI fibers were put on the wounds (Figure 5A). To visually observe the wound healing effect of dressings, the wounds were captured at different time points (Figure 5B). All the wound sizes are significantly reduced and Au_AI fibers can heal the wound faster than the other two groups. Statistical analysis shows the size of Au_AI fibers-treated wound is much smaller than those of gauze and PLGA fibers treated wounds, indicating the healing degree of Au_AI fibers group is obviously better than other two groups (Figure 5A). To analyze histology of the infected wound, the wounds tissues are stained with hematoxylin and eosin (H&E) and masson. After 6 days, the inflammatory cells (like neutrophils and lymphocytes) appeared, indicating the occurrence of inflammation (Figure 5C).54 The wound treated with Au_AI fibers group appears epithelial layer, indicating that the re-epithelialization of Au_AI fibers group is better than the other two groups.55 After 12 days, the inflammation in Au_AI fibers group faded away and there also appears a large number of fibrous cells and elongated fibroblasts which are beneficial to the formation of mature fibrous granulation tissue (Figure 5C).56 Compared with gauze and PLGA fibers, Au_AI fibers can reduce inflammatory reactions and accelerate the process of wounds healing. Masson's trichrome staining shows the formation and distribution of collagen in wound healing process.57 On day 6, Au_AI fibers treated wounds possess more collagen fibers (deeper blue color) than the other two groups (Figure 5C). On day 12, the distribution of

19



collagen become homogeneous and extend to the epidermis, indicating that the wound of the Au_AI fibers group heals up (Figure 5C).57 The Au_AI fibers can promote collagen deposition and epithelial formation, implying that Au_AI fibers as wound dressing to cure MDR bacterial wound infection are superior to gauze and PLGA fibers.

4. CONCLUSIONS We report a kind of non-antibiotic indole derivatives-capped gold nanoparticles (Au_IDs) with antibacterial activities against Gam-negative bacteria and even MDR bacteria. Au_IDs have excellent bactericidal activities eliminating most of the MDR bacteria even at high bacterial concentration which are superior to traditional antibiotics. Au_IDs fibers could effectively cure MDR bacterial infection as wound dressing in vivo. This work can broaden not only indole derivatives’ application, but also Au NPs-based antibiotic screening space, and may provide a prospective method for treating MDR bacterial infections.

APPROPRIATE FORMAT Supporting Information Characterization and antibacterial assays of Au_W and Au_AI, the synergistic effect between Au NPs and IDs against bacteria, optical density, bactericidal efficiency, colony assays, cototoxicity, SEM images of PLGA and Au_AI fibers.

AUTHOR INFORMATION 20


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Corresponding Author *Email: [email protected]. *Email: [email protected]. ORCID Chuanxin Ma: 0000-0001-5125-7322 Yukui Rui: 0000-0003-2256-8804 Xingyu Jiang: 0000-0002-5008-4703 Author Contributions ‡ These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the Chinese Academy of Sciences (121D11KYSB20170026) and the National Science Foundation of China (81361140345, 21535001, 81730051, 21761142006) for financial support.

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Figure and Table Caption:

Figure 1. Schematic illustration of the synthesis of Au_IDs and in vivo application in wound healing.

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Table 1. Antibacterial activities of Au_IDs and IDs against E. coli indicated with MIC (minimal inhibitory concentration, µg/mL).

MIC [µg/mL]

MIC [µg/mL]

IDs

IDs Au_IDs

IDs

Au_IDs

IDs

ID

7

>128

5-BI

15

>128

W

4

>128

5-CLI

30

>128

4-AI

8

>128

IAA

24

>128

5-AI

4

>128

5-CNI

13

>128

6-AI

13

>128

4-HI

17

>128

7-AI

11

>128

3-SI

>50

>128

32


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Figure 2. Characterization of Au_W and Au_AI NPs. (A) The UV-Vis spectra of Au_W and Au_AI (Inset: the optical images of Au_W and Au_AI). (B) The TEM images and particle size distribution charts of Au_W and Au_AI (d: diameter, f: frequency). (C) The FTIR spectra of W, Au_W, AI and Au_AI. (D) The XPS spectra of Au_W and Au_AI. (E) The NMR spectra (in DMSO-d6) of W, Au_W, AI and Au_AI.

33



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Table 2. Antibacterial/Bactericidal Activities Indicated with MIC (minimal inhibitory concentration, µg/mL)/MBC (minimal bactericidal concentration, µg/mL). MIC [µg/mL] / MBC [µg/mL] NPs

antibiotic-sensitive strains

clinical MDR isolates

E. coli

K. p

P. a

MDR E. coli

PR E. coli

Au_W

4/8

2/4

4/16

2/4

2/16

4/16

4/8

4/16

Au_AI

4/8

2/4

4/16

2/8

2/8

4/16

4/8

4/8

34


MDR K. p

PR K. p

MDR A. b

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Figure 3. The bactericidal activities of Au_W and Au_AI. (A) Time-dependent killing of MDR E. coli and PR K. p treated with Au_IDs and two antibiotics (polymyxin B and cefotaxime) (bacterial concentration: 2×105 CFU/mL). (B) Time-dependent killing of MDR E. coli and PR K. p treated with Au_IDs and two antibiotics (bacterial concentration: 5×108 CFU/mL). (C) Au_W and Au_AI induce permeability of cell membranes via STYO 9 (Green, viable cell) and propidium iodide (Red, dead cell).

35



Figure 4. The SEM and TEM images of E. coli treated with Au_W and Au_AI.

36


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Figure 5. The application of Au_IDs for accelerating wound healing. (A) The top images show the wound model under the treatment of different dressings. The bottom image shows wound size in different treatment groups at different observation time points. (B) Photographs of the wounds at different time points. (C) Histological graphs of skin tissue by H&E and Masson staining (Ly, lymphocyte; Ne, neutrophil; Ec, epithelial cells; Ef, elongated fibroblasts).

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Table of contents (TOC)

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Indole Derivatives-Capped Gold Nanoparticles as Effective

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