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Recyclable Escherichia coli specific-killing AuNP-polymers (ESKAP) nanocomposites Yuqi Yuan, Feng Liu, Lulu Xue, Hongwei Wang, Jingjing Pan, Yuecheng Cui, Hong Chen, and Lin Yuan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b02074 • Publication Date (Web): 20 Apr 2016 Downloaded from http://pubs.acs.org on April 24, 2016

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ACS Applied Materials & Interfaces

Recyclable Escherichia coli Specific-Killing AuNPPolymers (ESKAP) Nanocomposites Yuqi Yuan, Feng Liu, Lulu Xue, Hongwei Wang,* Jingjing Pan, Yuecheng Cui, Hong Chen and Lin Yuan* The Key Lab of Health Chemistry and Molecular Diagnosis of Suzhou, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou 215123, P. R. China. KEYWORDS: Escherichia coli, gold nanoparticle, specific-killing, glycopolymer, antibacterial polymer. ABSTRACT: Escherichia coli (E. coli) plays a crucial role in various inflammatory diseases and infections that pose a significant threats to both human health and the global environment. Specifically inhibiting the growth of pathogenic E. coli is of great and urgent concern. By modifying gold nanoparticles (AuNPs) with both poly(2-(methacrylamido)glucopyranose) (pMAG) and poly(2-(methacryloyloxy)ethyl trimethylammonium iodide) (pMETAI), a novel recyclable E. coli specific-killing AuNP-polymers (ESKAP) nanocomposite is proposed in this study, which based on both the high affinity of glycopolymers towards E. coli pili and the merits of antibacterial quaternized polymers attached to gold nanoparticles. The properties of nanocomposites with different ratios of pMAG to pMETAI grafted onto AuNPs are studied. With a pMAG:pMETAI feed ratio of 1:3, the nanocomposite appeared to specifically adhere to

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E. coli and highly inhibit the bacterial cells. After the addition of mannose, which possesses higher affinity for the lectin on bacterial pili and has a competitive advantage over pMAG for adhesion to pili, the nanocomposite was able to escape from dead E. coli cells, becoming available for repeat use. The recycled nanocomposite remained good antibacterial activity for at least three cycles. Thus, this novel ESKAP nanocomposite is a promising, highly effective and readily recyclable antibacterial agent that specifically kills E. coli. This nanocomposite has potential applications in biological sensing, biomedical diagnostics, biomedical imaging, drug delivery, and therapeutics. 1.

INTRODUCTION Various inflammatory diseases and infections are caused by bacteria such as pathogenic

Escherichia coli (E. coli); such diseases and infections greatly threaten public health.1-3 E. coli is one of the main species of pathogenic bacteria that reside in human and animal digestive tracts, and it causes various types of damage and pathological changes in the gastrointestinal and urinary systems.4,5 For example, a pathotype of E. coli known as uropathogenic E. coli (UPEC) causes serious urinary tract infections (UTIs), the most common and difficult-to-treat E. coli infection worldwide.4,6-8 The design of highly efficient drugs or approaches to treat diseases caused by E. coli has always been a topic of interest.3,9-11 Traditional therapies using small organic antibacterial agents or antibiotics are effective but have significant drawbacks, including large dosage requirements and lack of specificity. Moreover, such traditional therapies often facilitate drug resistance after long term and repeated use.12-16 Traditional treatments also disturb the balance of normal intestinal bacterial flora and may even damage the immune system, leading to secondary infections and poor therapeutic efficiencies.17-19 Therefore, the design of


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promising agents with strong bactericidal effects at lower dosages and with accurate targeting is critical to protect humans and the environment from pathogenic E. coli. In infections by most E. coli subtypes, fimbriae (or pili), long proteinaceous appendages, play an important role in the early stages of infection. These appendages act as a main virulence factor, mediating adherence to host cells as well as multiplication and dissemination, causing diverse diseases according to their pathotype.20,21 The interaction between bacteria and host cells is largely determined by the mannose-binding lectin FimH on the tips of the bacterial fimbriae. FimH mediates the adhesion of fimbriated bacteria specifically to oligosaccharide-containing receptors on the host cells.22-24 Because this protein has been shown to be involved in host cell recognition and to serve as a receptor for a specific target ligand, FimH is a potential molecular target for the design of therapies, drugs and detection methods.25-27 For example, monosaccharides or monosaccharide derivatives could be used as FimH inhibitors, binding to E. coli and blocking the adhesion of FimH to host cells.28,29 To enhance binding efficiency, monosaccharide-modified nanoparticles were created. Because nanoparticles have large surface area-to-volume ratios, they can carry more inhibitors. Multiple ligands on nanoparticles were found to increase nanoparticles interactions with E. coli.30,31 This strategy has great potential for the development of E. coli-specific killing materials, as antibacterial agents can be carried on nanoparticles. However, to ensure that the monosaccharides and antibacterial agents are free for binding, a longer covalent linker attachment to the nanoparticles may be necessary. Recently, it was reported that synthetic glycopolymers containing long chains and multiple glycol-based monomers were more effective than monosaccharides at binding E. coli. This increased binding affinity was mainly due to the glycocluster effect. Each monosaccharide in the glycopolymer is a pendant from the main chain, and it ensures that each monosaccharide is in a


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free state to bind with FimH. Moreover, polymer-immobilized nanoparticles have more physical and chemical properties, as well as extended applications.32,33 Such polymer-immobilized nanoparticles contain enough space to carry bioactive molecules while maintaining activity.34,35 For instance, gold nanoparticles modified with glycopolymers have obvious advantages, including specific recognition,36 binding ability,37 and colloidal stability,38 which show increased affinities for the lectins on red blood cells and improved adhesion to FimH on E. coli.39,40 Therefore, nanoparticles functionalized with glycopolymers and antibacterial agents can bind to E. coli specifically and then efficiently kill the attached bacteria. In this paper, we proposed a novel recyclable E. coli specific-killing AuNP-polymers (ESKAP) nanocomposite based on both the high affinity of glycopolymers towards E. coli pili and the merits of antibacterial quaternized polymers attached to gold nanoparticles.

In

this

new

strategy,

the

glycopolymer

poly(2-

(methacrylamido)glucopyranose) (pMAG) acts to specifically recognize the E. coli pili protein while poly(2-(methacryloyloxy)ethyl trimethylammonium iodide) (pMETAI) acts as the antibacterial agent. Therefore, targeting specificity can be increased, and the dosage of antibacterial agents can be reduced to a low level. In this work, the antibacterial activity, recyclability and the biocompatibility of particles prepared with different feed ratios of the two polymers were investigated and evaluated. Nanocomposites exhibiting high antibacterial activity and good biocompatibility were created. Most importantly, nanocomposite recycling can be achieved via elution with mannose, which helps the nanocomposite escape from dead E.coli cells and makes the material available for reuse. E. coli, as one of the main species and widely distributed groups of bacteria in the environment causing many infectious diseases,41 is worthy of profound investigation for


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the purpose of protecting humans and the environment. The novelty of this research is to provide a potential strategy of specifically and high-efficiently killing of accurate target E. coli in the environment without disturbing cell growth and unbalancing the normal bacterial flora, which are the troubles and possible difficulties in traditional therapy of infections and clinical practice. Meanwhile, by taking advantage of the small size of the nanoparticle, the multivalent effect of the glycopolymer, the high efficiency of the antibacterial agent and the competition of mannose, these ESKAP nanocomposites could realize not only the very strong bactericidal effects at much lower dosages, but also the recyclability of the particles; and these promise it a good biocompatible and environmentfriendly “green” antibacterial materials for wide applications in the fields of biomedicine, biosensors and environmental engineering. 2.

MATERIALS AND METHODS Materials Methyl iodide and hydrogen tetrachloroaurate hydrate were purchased from Sinopharm

Chemical Reagent Co. Ltd (Shanghai, China). Sodium citrate dihydrate (≥ 99 %), 2(dimethylamino)ethyl methacrylate (98 %), DL-dithiothreitol (≥ 99.5 %) and 5-aminofluorescein were purchased from Sigma-Aldrich. Methacryloyl chloride (stabilized with MEHQ, > 80 %) and D-(+)-glucosamine hydrochloride (> 98 %) were purchased from TCI. Agar powder and dialysis membranes (molecular weight cutoff (MWCO): 8-12 kDa) were obtained from Solarbio Science & Technology Co., Ltd. All bacterial growth media (tryptone, yeast extract and nutrient broth) were bought from Oxoid, and all animal cell culture media were purchased from Hyclone and Gibco. All other solvents and reagents were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China).


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Synthesis of 2-(Methacrylamido)glucopyranose (MAG) 2-(Methacrylamido)glucopyranose is the analogue of glucose. The synthesis of MAG was carried out according to a previously reported procedure with slight adjustments.42 Briefly, glucosamine hydrochloride (5.0 g, 23.2 mmol) and potassium carbonate (5.0 g, 36.2 mmol) were vigorously stirred in 150 mL of anhydrous methanol in a 250 mL single neck round-bottom flask in an ice bath (-10 °C) for 20 min, followed by the dropwise addition of methacryloyl chloride (2.18 g, 20.4 mmol) with vigorous stirring under a methanol/ice bath. The reaction was carried out for 4 h before being filtered through a sintered funnel with vacuum suction. The filtrate was then collected and concentrated to about 15 ml under vacuum. The products were purified via column chromatography using dichloromethane/methanol (ratio 4:1) as the eluent. The structure of the monomer was confirmed by 1H NMR analysis (Supporting Figure S3a). 1H NMR (300 MHz, D2O) δ 5.67 (s, 1H), 5.44 (s, 1H), 5.19 (d, J = 3.5 Hz, 0.57H), 4.73 (s, 0.43H), 4.03-3.66 (m, 3H), 3.57 (d, J = 9.6 Hz, 2H), 3.51 - 3.32 (m, 4H), 1.90 (s, 3H). Synthesis of Terminally Thiolated pMAG pMAG

was

synthesized

via

RAFT

polymerization

with

4-cyano-4-

(phenylcarbonothioylthio)pentanoic acid (CPADB) as the chain transfer agent (CTA) and AIBN as the initiator in a feed ratio of 200:1:0.5.43,44 A round-bottom flask was charged with MAG (0.471 g, 3 mmol), CPADB (0.004 g, 0.015 mmol), and AIBN (0.001g, 0.008 mmol) with 5 mL of DMF as the solvent. The sealed reaction vessel was purged with nitrogen for 30 min. The polymerization was carried in a preheated oil bath at 70 °C under a nitrogen atmosphere in a glove box. After 14 h, the polymer was dialyzed (MWCO: 8-14 kDa) against distilled water for 2 days. The structure of the polymer pMAG was confirmed by 1H NMR analysis (Figure 1c). 1H


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NMR (400 MHz, Deuterium Oxide) δ 5.14 (d, J = 44.8 Hz, 1H), 3.87 (s, 4H), 3.74 (d, J = 5.6 Hz, 2H), 3.49 (s, 4H), 1.75 (d, J = 107.2 Hz, 2H), 1.08 (s, 3H). For synthesis of terminally thiolated pMAG, the terminal groups of pMAG were further reduced via an aminolysis reaction by treated with an excess amount of 2-aminoethanol. After that, the solution was dialyzed for 1 day to remove the side products, and then lyophilized. The structure of terminally thiolated pMAG (pMAG-SH) was determined by 1H NMR (Supporting Figure S3b), and the molecular weight was measured with a Waters 1515 gel permeation chromatography system consisting of an Agilent PL Aquagel-OH column (8 µm, 300 × 7.5 mm). The mobile phase consisted of 1 L of Millipore water containing 0.2 mol L-1 NaNO3 and 0.1 mol L-1 NaH2PO4 mixed with 428.6 mL of anhydrous methanol. The mobile phase flow rate was 0.1 mL min-1. Synthesis of Terminally Thiolated Poly (2-(methacryloyloxy)ethyl trimethylammonium iodide) (pMETAI) pDAMEMA

was

synthesized

via

RAFT

polymerization

with

4-cyano-4-

(phenylcarbonothioylthio)pentanoic acid (CPADB) as the chain transfer agent (CTA) and AIBN as the initiator according to the approach reported by Mei et al.44 A feed ratio of 100:1:0.5 (monomer: CPADB: AIBN) was used. The polymer pDAMEMA was analyzed by 1H NMR in CDCl3 (Supporting Figure S3c) and by GPC in THF (Supporting Table S1). Cationic quaternized pDMAEMA (pMETAI) was obtained by following the procedures as described.45 The whole process was carried out in the dark. First, pDMAEMA (0.1 mmol) was dissolved in THF (50 mL) and kept stirring in an ice bath (-10 °C) for 30 min. Then it reacted with 5 molar equivalents of CH3I (0.5 mmol) for 3 h. After that, the pink precipitate was filtered out, and the solid was washed with n-hexane at least 3 times. The structure of the polymer pMETAI was confirmed by


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H NMR analysis (Figure 1d). 1H NMR (400 MHz, D2O) δ 4.54 (s, 2H), 3.89 (s, 2H), 3.32 (t, J =

8.4 Hz, 9H), 1.96 (d, J = 81.5 Hz, 2H), 1.10 (d, J = 52.1 Hz, 3H). For synthesis of terminally thiolated pMETAI, the terminal groups of pMETAI were further reduced via an aminolysis reaction by treated with an excess amount of 2-aminoethanol. Preparation of Citrate-Protected AuNPs Citrate-protected AuNPs were prepared as described previously.46 Briefly, Millipore water (100 mL) and HAuCl4 (12 mmol L-1, 516 µL) were mixed up in a 250 mL round-bottom flask equipped with a condenser, and heated to boil, then a sodium citrate solution (10% w/v, 4.4 mL) was poured into the reaction vessel with continuous stirring, resulting in color change from gray to burgundy. Boiling was kept for another 10 min, and stirring was continued until the mixture cooled to room temperature. It should be noted that all glassware in the synthesis of AuNPs AuNPs was cleaned with aqua regia solution (HCl/HNO3 = 3:1, v/v) and rinsed thoroughly with double deionized water prior to use. Preparation of Polymer-Immobilized AuNPs Terminally thiolated pMAG and pMETAI were both dissolved in Millipore water to final concentrations of 2 mg mL-1. Prepared AuNPs were centrifuged at 8000 rpm at 4 °C for 30 min, and the wash procedure was repeated 3 times. The polymer solution was mixed at different feed ratios and injected directly into the AuNPs colloid solution. The total volume of polymer solution was set at 40 µL for 1 mL of AuNPs colloid solution, and pMAG: pMETAI feed ratios were set to 1:1.5, 1:3 and 1:5 by volume. The immobilizing process was conducted in a shaker for 24 h at room temperature. After immobilization, the nanocomposite was centrifuged and washed at 8000 rpm several times to remove any excess polymer.


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Characterization of the Polymer Immobilized AuNPs The surface morphology of AuNPs was analyzed by transmission electron microscopy (TEM) (G-200, Hitachi, Tokyo, Japan), for which 10 µL of the sample was dried on the copper grid. Size measurements were performed at 25 °C via dynamic light scattering (DLS) with a Zeta sizer Nano-ZS90 zeta potential analyser (Malvern Instruments, UK). The interactions between the polymers and the AuNPs was investigated by spectrophotometry (Varioskan Flash, Thermo Scientific, USA). Synthesis of α-Thiol ω-Fluorescein pMAG and pMETAI The synthesis of α-pyridine disulfide ω-fluorescein pMAG and pMETAI were carried out according to the procedure described by Zhang et al.47 Approximately 3 equivalents of 2, 2dithiodipyridine were used to protect the α thiol group. Then, 5 equivalents of fluoresceinamine were conjugated to the ω-COOH group; this was followed by deprotection with DTT. The maximum absorption and emission wavelengths of the prepared polymers were determined using a Varicon Flash. Different pMAG-SH:pMETAI-SH-Fluo and pMAG-SH-Fluo:pMETAI-SH feed ratios were assembled onto the surfaces of gold nanoparticles, and the molar ratios of the two polymers on those surfaces were determined. The fluorescence intensities of the prepared nanocomposites at 4 nmol L-1 concentrations were determined. Bacterial Cultures E. coli (K-12) and Staphylococcus aureus (S. aureus) were obtained from the China General Microbiological Culture Collection Center (Beijing, China). The culture medium for E. coli consisted of peptone (4 g), yeast extract (2 g), NaCl (4 g) and 400 mL of Millipore water. The


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culture medium for S. aureus consisted of beef extract (4 g) and 400 mL of Millipore water. Each bacterial strain was cultured in sterile medium for 14 h in a shaker at 37 °C. Determination of Minimum Inhibitory Concentration (MIC) The minimum inhibitory concentration (MIC) of the prepared nanocomposites was determined according to the guidelines set by the Clinical and Laboratory Standards Institute (CLSI).48 The prepared nanocomposites were centrifuged and concentrated to yield a 30 nmol L-1 stock solution. Then, dilutions were performed to generate 1.2, 1.8, 3, 5, 6, 8, 10, 12, 15, 18 and 24 nmol L-1 solutions. Bacteria was grown to densities of 2 × 105 cells per milliliter. After incubation at 37 °C for 18 h, the optical densities of cultures were determined. Inhibition Experiment P4 and P5 were chosen for inhibition tests in the presence mannose. Briefly, the prepared nanocomposites were centrifuged at 8000 rpm for 15 min and dispersed in 100 µg mL-1, 200 µg mL-1, 500 µg mL-1 and 1 mg mL-1 mannose solutions. The concentration of nanocomposites was kept at 24 nmol L-1 in all solutions. After incubation at 37 °C for 2 h, 100 µL of the bacterial suspension was spread onto an agar plate and cultured for 14 h. Photos were taken to record the growth of the bacterial colonies, and colonies were counted by Image Pro Plus 6.0. Regeneration and Recycling of Nanocomposites The recyclability and regeneration capabilities of P4 were investigated. The particles were recycled a total of three times. The bactericidal abilities of the particles were assessed after each cycle. E. coli in early logarithmic phase with a density of 2 × 105 cells per milliliter was mixed with a 30 nmol L-1 solution of nanocomposites. After incubating for 2 h, the bacteria were separated by centrifugation at 6000 rpm for 3 min. The nanocomposites were separated via


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centrifugation at 8000 rpm for 15 min, then redispersed and treated with bacteria once again. Following each treatment, a 100 µL aliquot of the bacterial suspension was spread onto an agar plate. Photos of plates were taken after incubation for 16 h. Cytotoxicity Assay with L929 Cells Cytotoxicity against L929 was evaluated according to the MTT method.49 The prepared nanocomposites were centrifuged and dispersed in 1640 culture medium at a concentration of 12 nmol L-1. L929 cells were placed into 96-well plates at a density of 6000 cells per well. Nanocomposites were added to wells at final concentrations of 1 nmol L-1, 2 nmol L-1, 4 nmol L1

and 6 nmol L-1, with three replicates of each concentration. After culturing for three and five

days, the wells were washed with fresh culture medium and filled with 20 µL of MTT solution (20 mg mL-1). Any formazan that was formed was dissolved with DMSO after 3 hours of incubation. The absorption was measured at a wavelength of 490 nm. 3.

RESULTS AND DISCUSSION Preparation and Characterization of Polymer-Immobilized AuNPs (pMAG-pMETAI-

AuNPs) ESKAP nanocomposites were fabricated by grafting both pMAG and pMETAI polymers onto gold nanoparticles through Au-S bond after aminolysis (Scheme 1). First, we prepared gold nanoparticles via a sodium citrate reduction method. As expected, the gold nanoparticles had uniform spherical shapes with a narrow size distribution (Figure 1a and 1b). The mean diameter of the particles was approximately 14 nm.


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Scheme 1. (a) Preparation of the nanocomposites and their characteristics. Nanocomposites were fabricated by grafting both pMAG and/or pMETAI polymers onto gold nanoparticles. The bacterial binding and killing properties of the nanocomposites could be controlled by the feed ratios of the two polymers. The nanoparticle only grafted with pMAG (pMAG-AuNP) could achieve specific binding to E. coli. The nanoparticle only grafted with pMETAI (pMETAI AuNP) could kill E. coli in a non-specific way. The nanoparticle (ESKAP) both grafted with pMAG and pMETAI (pMAG-pMETAI-AuNP) could realize the specific killing of E. coli. (b) Synthesis route of the ESKAP. First pMAG and pDAMEMA was both synthesized via RAFT polymerization. pDAMEMA was then cationic quaternized

to pMETAI by CH3I. Both

terminally thiolated pMAG and pMETAI were obtained through an aminolysis reaction by 2aminoethanol. Finally, ESKAP nanocomposites were fabricated by grafting both pMAG and pMETAI polymers onto gold nanoparticles through Au-S bond.


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Figure 1. (a) TEM characterization of AuNPs. (b) DLS characterization for the diameters of AuNPs and nanocomposites immobilized with p1 (pMAG), p2 (pMETAI) and p1&p2 at different feed ratios (1:1.5, 1:3, 1:5 respectively for P3, P4 and P5). (c) 1H NMR spectra of pMAG. (d) 1H NMR spectra of pMETAI. Next, pDMAEMA and pMAG were synthesized via RAFT polymerization with CPADB as the chain transfer agent. According to GPC results (Supporting Table S1), the molecular weight was well-controlled. The tertiary amine groups in the pDMAEMA macromolecule chains were further quaternized with methyl iodide (CH3I) to generate pMETAI (Scheme 1b), a cationic quaternized pDMAEMA.45 The structures of both pMAG and pMETAI were proved by 1H NMR (Figure 1c and 1d).


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After reduction of the terminal groups in pMETAI and pMAG using an aminolysis reaction, the polymers were functionalized with thiol groups on one ends (Scheme 1b). A well-mixed solution of these two polymers was fed to the gold nanoparticle colloid at different feed ratios, and the self-assembly process was allowed to occur for 2 days with constant shaking. The final prepared polymer-immobilized AuNPs (pMAG-pMETAI-AuNPs) were characterized with visible spectrometry and dynamic light scattering (DLS). In addition, the true molar ratios of the two immobilized polymers were calculated for different polymer feed ratios from the fluorescence intensity of fluoresceinamine, which was conjugated to terminal carboxyl groups via carbodiimide chemistry (Figure 2c). From the ultraviolet visible absorption spectra (Figure 2a), it was apparent that newly prepared gold nanoparticles exhibited an absorption maximum at 520 nm. After polymer immobilization, the absorption maximum of the nanocomposite was redshifted, indicating that the polymers were successfully grafted onto the surfaces of the gold nanoparticles. The modification of the polymers on AuNPs greatly changed the surface charge of the nanocomposites. From the zeta potentials (Figure 2b) of these materials, we observed that the nanocomposites grafted with only pMAG were negatively charged (-24.90 ± 0.65 mV), similar to the unmodified nanoparticles. In contrast, nanocomposites grafted with both polymers became positively charged (34.17 - 45.52 mV). This charge reversal could be attributed to the high positive charge of pMETAI. Recent researches had demonstrated that the introduction of new substance onto the surface of nanoparticles could bring new functions.50,51 Because the antibacterial activity of the quaternized polymers is due to positively charged groups in the structure, the positive charge on the surface of these nanocomposites might allow them to kill E. coli.


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Figure 2. (a) Visible spectra of the AuNPs and prepared nanocomposites. (b) DLS characterization for zeta potential of the AuNPs and prepared nanocomposites. (c) Table of the feed ratio, diameter distribution and quantitative analysis results of the molar ratio of the two polymers (p1: pMAG; p2 : pMETAI) on the nanocomposites. Evaluation of the Antibacterial Properties of ESKAP The antibacterial activity of the nanoparticles prepared with different pMAG to pMETAI feed ratios was investigated by colony counting. As shown in Figure 3a, the nanoparticles only grafted with pMAG (P1) did not exhibit any antibacterial activity. After both grafting with pMAG and pMETAI, the nanocomposite (ESKAP) was able to inhibit the growth of E. coli. Antibacterial activity increased as the amount of pMETAI was increased (Figure 3b). At a pMAG:pMETAI feed ratio of 1:1.5, the inhibition rate was 53.0 ± 6.4 %. At pMAG:pMETAI feed ratios of 1:3 and 1:5, the inhibition rate climbed as high as 98.3 ± 0.1 % and 98.6 ± 0.5 %. These results indicate that the antibacterial ability of the nanocomposite particles is due to


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pMETAI, a quaternary ammonium salt and a widely-used cationic fungicide. pMETAI can change the permeability of the cell membrane and inhibit bacterial metabolism.52

Figure 3. (a) Agar plates of E. coli at the density of 2 × 105 treated with P1, P3, P4 and P5 for 3h at the concentration of 12 nmol L-1; then the plates were incubated at 37 °C for 16h. (b) Inhibitory rate of the prepared nanocomposites according to the number of colonies on the agar plates, which were counted by Image Pro Plus 6.0. Data are the mean ± SD (n = 3). However, gold nanoparticles grafted with only biocidal pMETAI (P2) exhibited a much lower killing ability compared to P4 and P5, which were grafted with both pMETAI and pMAG (Figure 4). This result demonstrates that the pMAG glycopolymer plays an important role in the antibacterial properties of the ESKAP, and the addition of pMAG can significantly enhance antibacterial effects. Further quantitative evaluation of antibacterial activities was performed via standard gradient dilution and plating. The results from this evaluation indicated that the minimum inhibitory concentration (MIC) of the nanocomposite particles was approximately 3


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nmol L-1. It was markedly lower than pMETAI45 and might be due to high local concentrations of pMETAI on baterial cell wall resulted from the specific adhension of pMAG to FimH. The majority of E. coli strains possess type 1 fimbriae; these fimbriae are surface organelles that mediate specific binding to D-mannose-containing structures.53 Previous studies on the fimbriae of E. coli have found that FimH, a minor subunit of the tip fibrillum protein, is the determinant of the protein’s mannose-specific binding property. It has been reported that the FimH protein possesses carbohydrate recognition sites (CRS) with high affinity for mannose and glucose.54 So, it is presumed that the enhanced bactericidal effect of the ESKAP is due to the presence of the pMAG polymer, which improves the adherence of particles on the bacterial surface and enhances the antibacterial effect of pMETAI. The effect of pMAG on the adsorption capacity of the nanocomposite particles on bacteria was determined and proven by differential centrifugation fluorescence staining and SEM (Supporting Figure S1&S4). The enhancement in adherence might be due to specific recognition of the lectin protein and the multivalent effect of pMAG.


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Figure 4. Bacterial growth treated with P1, P2, P4 and P5 at the concentration of 12 nmol L-1 and further incubated at 37°C for 16 h. Data are the mean ± SD (n = 3).

Figure 5. (a) Numbers of E. coli colonies on the agar plates, which were previously treated with P4 and P5 (both at 24 nmol L-1) containing mannose as inhibitory agent at various concentrations (100 µg mL-1, 200 µg mL-1, 500 µg mL-1 and 1 mg mL-1). (b) Agar plates of the inhibitory experiment consistent with (a). To further demonstrate the positive effect of pMAG in ESKAP, mannose solutions at varying concentrations were introduced into the system. Because the carbohydrate recognition sites


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(CRS) of FimH possess higher affinities for mannose (Kd 2.3 mmol L-1) than for glucose (Kd 9.24 mmol L-1),54 mannose can compete with pMAG for adhesion to bacterial pili. This competition reduces the adhesion capacity of nanoparticles to bacteria, weakening the bactericidal capacity of the nanoparticles. As shown in Figure 5, after dispersal in the mannose solutions, the P4 and P5 complexes showed gradually decreasing E. coli killing abilities as mannose concentration was increased. After treatment with 100 µg mL-1 mannose, the bactericidal capacity of the P4 particles was greatly reduced. When the mannose concentration was to 1 mg mL-1, almost all of the bactericidal capacity of the nanocomposites was lost. These results demonstrate that competition between mannose and pMAG for binding to bacterial pili markedly reduces the specific adsorption capacity of the nanocomposite particles, reducing the effective local dose of biocide on the bacterial surface, and suppressing the antibacterial effect of the nanocomposites. Sustainable Recycling of ESKAP The lectin on the fimbriae in E. coli has a mannose-binding domain. This domain exhibits much greater affinity for mannose compared to glucose or other sugar-containing compounds. Theoretically, the nanocomposite, which has accomplished its bactericidal purpose but remains adhered to dead cells, can be washed off of E. coli cells via treatment with large amounts of mannose. Such washing should restore the bactericidal ability of the nanocomposites. To verify this hypothesis, we repeatedly recycled the nanocomposites. The results showed that the ESKAP nanocomposites had a high recovery efficiency and antibacterial effect: after 3 repeated uses, the nanocomposites retained their antibacterial efficiency almost completely (Figure 6). Recycling and reusing this antibacterial nanocomposite will help improve long-term sustainability and performance while reducing cost and environmental impact.


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Figure 6. (a) Recyclability study of P4, the initial concentration of which was set to be 30 nmol L-1 and all the nanocomposites were regenerated after being treated with E. coli at the density of 2 × 105. (b) Sketch of the recycle process. Effect on Gram-Positive Bacteria Gram-positive bacteria differ from Gram-negative bacteria in their cell wall architecture and many other features.55,56 In this study, the antibacterial effect of the nanocomposites on Grampositive Staphylococcus aureus (S. aureus), another serious human pathogen, was investigated the target specificity of this novel nanocomposite material. The results showed that the antibacterial efficiency of ESKAP nanocomposites on S. aureus was much lower than on E. coli. (Supporting Figure S2&S5). This difference is mainly due to the different binding abilities of ESKAP nanocomposites to these two types of bacteria. Glyco-sensitive fimbriae in E. coli mediate binding to receptor structures, allowing nanoparticles to aggregate and adhere to


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bacterial surfaces. Thus, even at very low doses of ESKAP, the local effective concentrations of antibacterial agents on the bacteria surface are high enough to permit full interactions between the antibacterial components and the bacteria, resulting in a strong bactericidal effect. In contrast, due to the absence of glycol-sensitive fimbriae in S. aureus, the pMAG on the nanocomposites cannot specifically bind to S. aureus cells, which leads to a poor bactericidal effect at low dosages. These results demonstrate that these novel ESKAP nanocomposites (pMAG-pMETAIAuNPs) have a selective and specific killing effect on E. coli cells. Biocompatibility Evaluation of ESKAP The good biocompatibilities of nano-gold materials have been widely reported. However, pMETAI is highly toxic to bacteria as well as normal animal cells. Therefore, by introducing pMAG into this novel nanocomposite material, it is expected that high-level and effective local dosages of antibacterial agents can be achieved on bacterial surfaces with minimal dosages. Reducing the amount of antibacterial agents used could increase the biocompatibility of this material.

Figure 7. (a) Light field images of L929 cells treated with P1, P2 and P4 at the concentration of 6 nmol L-1 after culturing for 3 days. (b) The viability of L929 cells cultured in the medium


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containing P1, P2 and P4 at different concentrations for 3 days through MTT test. Data are the mean ± SD (n = 3). To evaluate biocompatibility, varying concentrations of materials were applied onto L929 fibroblasts. Effects on fibroblast growth were investigated by methyl thiazolyl tetrazolium (MTT) cell viability analysis. The results showed that P2, which consisted only of grafted pMETAI, inhibited cell growth at the third day of treatment (Figure 7). From the light field images in Figure 7a, it could be seen that after treated with 6 nmol L-1 P4, the growth and the morphology of L929 cells were both quite good compared to control cells. When treated by even higher concentrations of P4 (12 nmol L-1), the growth of L929 cells was still similar with the control and almost all the cells were alive (Supporting Figure S6). The results indicated that ESKAP nanocomposites had significantly improved biocompatibilities. Its biocompatibility can be attributed to the introduction of glycopolymers onto gold nanoparticles as well as the low concentrations necessary to kill E. coli. Nowadays, functionalization of nanomaterials with novel physicochemical, biochemical, morphological, and functional properties has achieved a great of concerns. These advanced functionalized nanomaterials have opened up unlimited possibilities for nano-science and nanotechnology. When functionalized with specific substances, the well-controlled nanomaterials, which are endowed with specific target recognition and binding function, have been widely used for specific inorganic or organic small molecular targets in the applications ranging from biomedicine, industry and environment protection.50,51,57 For example, the multi-walled carbon nanotube (MWCNT) integrated with different functional factors could competent for various missions in waste water treatment aiming to specifically remove of arsenic and lead ions,58-60 adsorb chromium contaminants,61 and degrade rhodamine B.62 The excellent performances of


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these nanomaterials benefit from the combination of the high surface activities of the nano-sized materials and the distinct specific-targeting properties of the functional factors. This strategy can be well applied for more complicated systems, such as biological analysis and clinical treatment. In our work, the prepared ESKAP accumulates the advantages of the high loading property from gold nanoparticles, the high killing efficiency to E. coli from pMETAI, and the specific binding activity to FimH from pMAG. Moreover, the ratio of different functional polymers plays an important role in balancing the antibacterial and biocompatible effects. The study herein reveals a new approach in designing nanocomposites by introducing the polymers for environmental and biomedical research. Considering different affinity and sensitivity of various bacteria, the glycounits and antibiotic agents could be changed in order to effectively handle the complexity of bacteria in open environment.

4.

Conclusions In this work, a recyclable AuNP-polymer (ESKAP) nanocomposite capable of specifically

killing E. coli was fabricated by introducing quaternized pDMAEMA and pMAG onto gold nanoparticles using a facile one-step synthesis method. In this ESKAP nanocomposite, gold nanoparticles provide an excellent scaffold with excellent features, such as good biological compatibility, high stability, large surface area-to-volume ratios, and easy modifiability. The modification of quaternized pDMAEMA (pMETAI) not only endows the nanocomposite with remarkable bactericidal capacity, but it also generates a positively-charged protective layer on the surface of the AuNPs, which ensures nanoparticle stability in high salt solutions and in complex physiological conditions. Another important component of the ESKAP nanocomposite, pMAG, acts as a bridging molecule for specific adherence to E. coli bacteria. The specific


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affinity of the polymer for the pili of E. coli effectively improves the bacterial target specificity of the antibacterial material. Additionally, the multivalent effect of the glycopolymer strengthens the binding ability of gold nanoparticles with bacteria,43 thus enhancing the local concentration of biocidal agents on the bacterial surface and lowering the necessary drug dosage. Analysis of antibacterial effectiveness showed that the materials created in this work (pMAG-pMETAIAuNPs) exhibit significant advantages, especially low effective dosages and specific killing of E. coli, compared with gold nanoparticles grafted only with pMETAI. Furthermore, the results of the recycling experiment demonstrated that this material can be recycled and reused. Given the above advantages as well as the easy preparation and cheap cost of the material, the ESKAP nanocomposite (pMAG-pMETAI-AuNPs) developed in this work may find potential applications as a useful and E. coli-specific killing agent in the healthcare and environmental engineering industries.

Supporting Information The molecular weights and 1H NMR of polymers, the dissociation of ESKAP and E. coli by mannose, the effect of ESKAP on S. aureus and on the mixture of E. coli & S. aureus coculture, live/dead staining assays for L929 cells. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *

Tel:

+86-512-65880567.

Fax:

+86-512-65880583.

E-mail:

[email protected];

[email protected].


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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by the National Natural Science Foundation of China (21334004, 21374070 and 21474072), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD), and the Project of Scientific and Technologic Infrastructure of Suzhou (SZS201207). REFERENCES (1)

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Recyclable Escherichia coli-Specific-Killing AuNP ... - ACS Publications

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