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Functional Structure/Activity Relationships

Synthesis of Amphiphilic Copolymers Containing Ciprofloxacin and Amine Groups and their Antimicrobial Performances Revealed by CLSM and AFM Man He, Yuming Zhou, Shuangxi Nie, Peng Lu, Huining Xiao, Yuan Tong, Qiang Liao, and Ruili Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01759 • Publication Date (Web): 17 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Journal of Agricultural and Food Chemistry

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Synthesis of Amphiphilic Copolymers Containing Ciprofloxacin and Amine

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Groups and their Antimicrobial Performances Revealed by CLSM and AFM

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Man He,†,‡ Yuming Zhou,*,† Shuangxi Nie,‡ Peng Lu,‡ Huining Xiao,§ Yuan Tong,† Qiang Liao†

5

and Ruili Wang†

†

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†

School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China

‡

Guangxi Key Laboratory of Clean Pulp & Papermaking and Pollution Control, College of Light Industry

and Food Engineering, Guangxi University, Nanning 530004, China §

Department of Chemical Engineering & Limerick Pulp and Paper Centre, University of New Brunswick,

Fredericton, NB E3B 5A3, Canada

12 13

*Corresponding Authors:

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Tel.: +86-25-52090617; Fax: +86-25-52090617;

15

E-mail: [email protected] (Yuming Zhou)

16

1

ACS Paragon Plus Environment


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ABSTRACT: Two series of amphiphilic antimicrobial copolymers containing ciprofloxacin (CPF) and

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amine functional group have been synthesized via free radical copolymerization. The chemical structures

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of different amine groups and the copolymer composition have been systematically varied to study how

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structure of the copolymers exerted an influence on their antibacterial activity. The viability of Escherichia

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coli in the presence of antimicrobial copolymers was observed by confocal laser scanning microscopy

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(CLSM). CLSM as well as atomic force microscope (AFM) were applied to visualize changes in bacterial

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morphology treated with antimicrobial copolymers and discussed the antimicrobial mechanism of

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antimicrobial copolymers. Morphological changes of bacteria observed via AFM and CLSM demonstrated

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the antibacterial mechanism was due to the disruption of bacterial membrane. The destruction of the cell

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membrane was also confirmed by the leakage of intracellular components which had a strong absorbance

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at 260 nm. The inhibitory process was monitored by UV absorption dynamically.

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KEYWORDS: copolymers, antimicrobial activity, UV absorption, AFM, CLSM

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1.

INTRODUCTION

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Microbial contamination has attracted wide attention in various fields, such as medical devices, textiles,

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food package, and food storage.1,2 Antimicrobial agents3-7 have gained interest from both academic

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research and industry because of their potential to provide quality and safety benefits for many materials.

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But the antimicrobials with low molecular weight suffer from many shortcomings, such as short-term

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antimicrobial ability and environmental toxicity. In order to overcome these drawbacks, researchers have

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made great efforts to develop novel polymers with excellent antibacterial activities. Amphiphilic cationic

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polymers have attracted scientific interest because of the enhanced antibacterial activities of the

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polymers.8-12 The antibacterial activities of the amphiphilic polymers can be readily adjusted and controlled

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just by varying the proportions of hydrophobic and cationic monomers.13-18 Thus, a perfect amphiphilic

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balance from the optimum combination of cationic and hydrophobic monomers can achieve high

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antimicrobial activity.19-22 electrostatic attraction of the cationic groups to the bacterial membrane with

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negative charge causes destruction of the bacterial membrane, resulting in cell death.23-25

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In addition to high antimicrobial performances, a crucial feature from amphiphilic polymer research is that

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bacteria fail to develop resistance.26 Bacteria with disrupted bacterial membranes cannot reconstitute and

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therefore lose resistance against antimicrobial polymers.27 Here, a hydrophobic monomer and cationic

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monomers containing primary or quaternary amine groups were introduced into the polymers to impart the

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polymers amphiphilic properties, which were very important to exhibit antibacterial properties. The

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interplay between the structures of different amine functionalities and antibacterial properties was

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investigated to reveal the effect of amine functional groups on antimicrobial activities. The molar ratios of

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hydrophobic side chain and polymer composition were changed to survey the optimum hydrophilic and 3



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hydrophobic balance.

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Ciprofloxacin (CPF) is a widely used class of antimicrobials that inhibit bacterial duplication by acting on

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DNA gyrase. Because of effective bactericidal activity, the antibiotic ciprofloxacin was introduced into the

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copolymers to further enhance the antimicrobial properties of the copolymers and prepare a series of

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amphiphilic copolymers.

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Several techniques have been used to investigate antimicrobial mechanism and interactions between

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antibacterial copolymers and bacteria. Confocal laser scanning microscopy is capable of achieving the

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high-resolution images of microbial cells and has been widely applied in antimicrobial mechanism

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study.28-33 CLSM has been applied to analyse bacterial viability and observe bacterial morphology via

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staining microbes with special fluorescent substances. Atomic force microscope has become another very

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useful imaging instrument that enables high resolution imagings of biospecimens.34-36 AFM technique also

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allows investigator to probe antibacterial mechanism via revealing morphological transformation of

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microbes under antibacterials. The dynamic antibacterial process is monitored using UV because leakage

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of cellular contents from microbes shows absorption at 260nm. The use of UV absorption to measure

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release of cellular contents from microbes has become a popular method to investigate the antimicrobial

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dynamics of copolymers37-39 In this work, the antibacterial properties of antimicrobial copolymers were

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estimated by quantifying the minimal inhibitory concentration (MIC) values, which is minimum polymer

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concentration completely inhibiting bacterial growth. The viability and the morphological change of

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microbes after exposure with antimicrobial copolymers could be efficiently monitored by CLSM to

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explore the mechanism of interaction of antimicrobial copolymers with bacterial cells. The morphology of

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bacteria exposed to antimicrobial copolymers was observed by AFM and CLSM to reveal the antibacterial 4


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mechanism of antimicrobial copolymers. UV260 absorbance was used to monitor the dynamic antibacterial

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process.

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Compared to previous work,40,41 in this work, although the content of CPF in the copolymer was reduced,

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these antibacterial copolymers showed excellent antimicrobial activities due to perfect amphiphilic balance

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obtained by changing the molar ratio of monomers. The effect of different amine functionalities on the

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antibacterial properties was discussed in detail and the antibacterial mechanism was revealed by AFM and

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CLSM.

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2.

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2.1. Materials. Ciprofloxacin, Glycidyl methacrylate (GMA), 2-(Dimethylamino) ethyl acrylate methyl

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chloride (DMAEAMC), 2-Aminoethyl methacrylate hydrochloride (AEMAH), Luria Bertani (LB) agar

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and Phosphate-buffered saline (PBS) were all purchased from Aldrich. Escherichia coli (ATCC11229) was

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cultivated overnight at 37 ℃in LB broth before use. The monomer GMA-CPF was synthesized as the

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process previously described.40

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2.2. Synthesis of Copolymers with Primary Amines. Butyl acrylate and AEMAH (9.5 mmol total,

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various molar ratios) dissolved in 7 mL of DMF and GMA-CPF (0.2371 g, 0.5 mmol) were added to a 25

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mL round-bottom flask. Then AIBN (0.5 wt. % of the total monomer weight) as an initiator were added to

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the reaction mixture. The solution was deoxygenated with N2 bubbling and the flask was sealed with a

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rubber plug. The reactants was stirred at 65℃for 24 h and precipitated into diethyl ether to give sticky

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yellow product. The crude product was dialyzed against distilled water and the product was dried in

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vacuum. Copolymers containing primary amine groups are denoted as series A. 1H nuclear magnetic

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resonance (1H-NMR) (dimethyl sulfoxide DMSO, 300 MHz) for copolymer A6 (AEMAH: BA:

MATERIALS AND METHODS

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GMA-CPF=62:35.7:2.3): 0.76 (s, 131.93H), 1.01 (s, 4.35H), 1.18 (s, 31.40H), 1.35 (s, 31.82H), 1.51 (s,

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15.85H), 1.95 (s, 87.30H), 2.27 (s, 1.96H), 2.43 (s, 0.94H), 2.79 (s, 3.97H), 2.98 (s, 4.05H), 3.23(s,

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53.95H), 3.67 (s, 34.15H), 4.28 (s, 54.54H), 7.53 (s, 1.03H), 7.78 (s, 1.00), 8.65 (s, 0.96H). 1H-NMR

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spectrum of A6 (AEMAH: BA: GMA-CPF=62:35.7:2.3) copolymer is shown in Figure S3.

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2.3. Synthesis of Copolymers with Quaternary Ammonium Salts. The copolymers with quaternary

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ammonium salts were prepared according to the procedure described above for copolymers containing

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primary amines. The copolymers with quaternary ammonium salts were prepared via changing monomer

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molar ratio and denoted as series B. 1H-NMR (DMSO, 300 MHz, ppm) for the copolymer B7

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(DMAEAMC: BA: GMA-CPF=47.8:50:2.2): 0.87 (s, 71.20H), 1.15 (s, 4.65H), 1.38 (s, 45.50H), 1.55 (s,

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45.80H), 1.76 (s, 44.25H), 2.41 (bs, 90.50H), 2.78 (s, 1.95H), 2.91 (s, 1.02H), 3.15 (s, 195.60H), 3.44 (s,

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3.85H), 3.53 (s, 3.79H), 3.69 (s, 43.40H), 4.07 (s, 48.30H), 4.49 (s, 43.50H), 7.53 (s, 1.02H), 7.84 (s,

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1.00H), 8.67 (s, 1.03H). 1H-NMR spectrum of B7 (DMAEAMC: BA: GMA-CPF=47.8:50:2.2) copolymer

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is shown in Figure S4.

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2.4. Characterization of GMA-CPF and Copolymers. 1H-NMR and

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and DMSO-d6 using an Oxford spectrometer (300 MHz). A thermogravimetric analyzer (SDT-Q600) was

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used to get the thermogravimetric analysis curves of the copolymers with CPF and pure CPF under a dry

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nitrogen atmosphere and at 30 to 800 ℃.

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2.5. Characterization of Antimicrobial Activities. MICs of antibacterial copolymers were measured

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using the broth microdilution method. Cultures of E. coli were diluted to 106 CFU/mL with LB broth and

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used for antimicrobial tests. A copolymer solution was made in sterile deionized water and then diluted

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with LB broth to a constant volume. An equal volume of bacterial suspension was added into copolymer 6


13

C-NMR were performed in D2O

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broth solution. In the end, all the tubes were incubated overnight at 37 ℃.

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The use of UV absorption to measure leakage of cellular contents from microbes has become a popular

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method to survey the antibacterial dynamics of copolymers.37-39 E. coli suspension was centrifuged,

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washed with deionized water twice and redispersed with PBS. The same amount of E. coli was added to a

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few test tubes, which were each added with the desired amount of copolymer solution except the control.

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Then 1.5 mL sample was removed using a pipet from each tube and injected into a quartz cuvette. The

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sample absorption at 260 nm was monitored with a Genesys 10 UV-Vis spectrophotometer.

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AFM experiments were performed to study the morphological changes of E. coli untreated and treated by

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antimicrobial copolymers. Bacterial cells were dispersed on a silicon wafer and allowed to dry at room

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temperature. AFM measurements were performed with a Veeco Nanoscope ⅢA using tapping mode.

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CLSM was used to investigate the viability and the morphological changes of microbes treated with

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antimicrobial copolymers. A fresh working solution was prepared by the addition of 50uL of fluorescein

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isothiocyanate (FITC) dissolved at 5mg/mL in anhydrous alcohol, 40 uL of propidium iodide (PI)

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dissolved at 1mg/mL in PBS to 1.9 mL of PBS. Then 3 uL of a working solution which contained a certain

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amount of antibacterial copolymer was added to 1 ml of bacteria suspension. All microbes containing live

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and dead showed green fluorescence at 488nm excitation wavelength, while dead microbes displayed red

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fluorescence at 543nm excitation wavelength. The stained E. coli was applied on a glass slide and sealed

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with a cover slip for imaging with a Leica SP2 confocal microscope.

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3.

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3.1. Characterization of GMA-CPF and Copolymers. Figures S1 and S2 show 1H and 13C-NMR spectra

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of the methacrylate monomer GMA-CPF. Figure 1 shows that antimicrobial copolymers series A and B

RESULTS AND DISCUSSION

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containing different amine functional groups were prepared via a free radical polymerization of GMA-CPF,

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hydrophobic monomer BA and cationic monomers with ammonium groups by employing AIBN as the

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inhibitor at 65 ℃ for 24 h. Figures S3 and S4 demonstrate 1H-NMR spectra of copolymers A6 and B7.

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The double bonds peaks at 5.67 and 6.10 ppm for GMA-CPF disappeared upon polymerization. New

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signals are observed at 1.51 and 1.95 ppm, corresponding to the backbone proton of A6. Similarly, new

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peaks at 1.76 and 2.41 ppm after the polymerization were formed, corresponding to the backbone proton of

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B7. The structures of A6 and B7 were confirmed by 1H-NMR. The mole ratio of BA in the copolymers was

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adjusted by altering the feed ratio of comonomers. The copolymer components were identified from

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1

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B. Molecular weights of the cationic copolymers were tested by aqueous gel permeation chromatography

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(GPC). However, no results were found because cationic copolymers were chemisorbed on the GPC

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column with anionic packing beads. The molecular weights of the copolymers should be between 10,000

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and 15,000.42

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Thermal stability of the antimicrobial copolymers was assessed by thermogravimetry (TG; Figure 2). The

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TG curves between 30 and 800 ℃ suggest copolymers A4 and B4 degraded in two-steps, with initial step

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at 160-295 ℃ (weight loss due to amine groups) and final step at 295-470 ℃ (due to CPF). Pure CPF

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decomposed in a single-step at 285-470 ℃, which coincided with the second degradation of the

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copolymers. The antimicrobial copolymers can be applied in many fields, such as functional fabrics and

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water system due to their heat stability.

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3.2. Antimicrobial Effects of Copolymers. With the MIC method, antibacterial properties of the

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copolymers were evaluated against Gram-negative E. coli (Tables 3 and 4). Series A contained a similar

H-NMR (Tables 1 and 2). The BA mole ratio ranged from 9.8 to 35.7 in series A and 21.5 to 50.0 in series

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mole ratio of GMA-CPF but showed various MICs via adjusting the mole percentages of AEMAH and BA

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(Table 3). Similarly, series B containing a similar mole percentage of GMA-CPF had various mole

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percentages of DMAEAMC and BA as well as various MICs (Table 4). The structure-activity relationships

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of the antimicrobial copolymers were investigated to reveal the effect of different amine functionalities and

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hydrophobic group on antimicrobial abilities. The MICs of copolymers A and B depended on the BA

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content (Figure 3) and decreased as BA content increased, indicating the increased hydrophobicity of side

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chains improves antibacterial properties. The MICs of series A showed a minimum of 4 ppm as BA content

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varied within 27.4-35.7. In series B, when the BA content reached 39.0 the MICs minimized to 7.8 ppm.

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There were two main processes that contributed to the antibacterial property of the amphiphilic

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copolymers. Firstly the copolymers with positive charge were driven by electrostatic interaction to attack

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the cell membranes with negative charge. The copolymers displaced cations (e.g. Ca2+ and Mg2+) within

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the membrane and may lead to a loss of membrane integrity.43 Secondly, the hydrophobic side chains of

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the amphiphilic copolymers inserted into the hydrophobic region of the cell membrane, resulting in

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membrane rupture and the release of intracellular components.44 An optimum hydrophobicity of the

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amphiphilic copolymers allowed them to enter and diffuse in the lipid membranes of Gram-negative

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bacteria. In this study, as both copolymers A and B became more hydrophobic, and were more potent to

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permeabilize membranes by insertion of hydrophobic groups and thus disrupted bacterial cell membranes.

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Hence, the increased hydrophobicity of the copolymers led to antibacterial improvement due to the

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enhanced membrane binding and permeablization.

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In addition, the relationship between the structures of amine functionalities and antimicrobial properties

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was investigated to reveal the antibacterial abilities of the copolymers were profoundly affected by amine 9



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functionalities. When the BA contents in copolymers A and B were similar, copolymers with primary

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amine side chains demonstrated lower MICs compared to copolymers with quaternary ammonium side

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chains (Figure 3). The results indicated the primary amine groups increased antibacterial activities, while

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the copolymers containing quaternary ammonium side chains were less potent to inhibit bacterial growth.

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Cationic charge and chemical structure of the ammonium groups both were crucial on antibacterial activity.

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The bactericidal mechanism of copolymers may involve complexation of ammonium groups to anionic

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Under this circumstance, the hydrogen bonds may be formed between the protonated ammonium groups

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and lipids and thus the copolymers containing primary amine groups demonstrate stronger antibacterial

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properties than those containing quaternary ammonium groups.16 The copolymers with quaternary

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ammonium salts need more hydrophobicity to obtain extensive antibacterial potency in comparison with

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the copolymers with primary amine side chains.16

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The BA content of copolymer B8 was similar to that of copolymer B1 and the CPF mole content in

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copolymer B8 was twice that in copolymer B1. With the increase of CPF content, the MICs of the

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antimicrobial copolymers declined, indicating CPF considerably affected the antimicrobial capability, in

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which CPF is absorbed into the nuclear region and inactivates DNA gyrase, thus killing bacterial cells.

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Although the bactericidal activity of CPF is well known, the bactericidal mechanism of CPF moieties in

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the copolymers remains unclear, and the antibacterial activity of the CPF moieties in the copolymers

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cannot be explained. Some possible mechanisms have been proposed, involving either the absorbance of

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copolymers into the nuclear region or the breakage of CPF from the copolymers. Because the large sizes

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restricted the spread of the copolymers via the cell membranes, the breakage and spread of CPF were more

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liable than the spread of the copolymers into the bacteria.45 The hydrolysis of the ester bonds may cause 10


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small-molecule CPF to release.

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3.3. Antimicrobial Mechanism of Copolymers. The UV absorbance at 260 nm underlies the mechanism

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concerning the interaction between bacterial cells and the antimicrobial cationic copolymers. The release

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of intracellular parts, especially DNA and RNA (with strong absorption at 260 nm), can be monitored by

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UV. Figure 4 shows the UV-vis spectrum on the release of materials absorbing 260 nm upon addition of

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antimicrobial cationic copolymers to E. coli suspensions, and the ratio of O.D. of bacteria suspensions with

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versus without antimicrobial copolymers plotted versus time. In E. coli suspensions treated with

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antimicrobial copolymers at 50 ppm (> MIC), the OD260 increased for 10 minutes (Figure 4A). This quick

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release of the 260 nm absorbing materials agreed well with rapid killing kinetics of the antimicrobial

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copolymers. Owing to a dominant membrane active mode, the antimicrobial copolymers were able to kill

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bacterial cells within minutes at the concentration above MIC. The final OD260 for copolymer A4 at above

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MIC was far larger than for copolymer B4 at below MIC (Figure 4B). Results showed the antimicrobial

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copolymers exerted potent bactericidal effects at above MIC. The 260 nm absorbing materials was released

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from the bacterial cytoplasm which had no outer cell membranes.

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To explore how antimicrobial cationic copolymers affected the morphology of bacterial cells, we took

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AFM topography images, height images and section images of E. coli before and after treatment with

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antimicrobial copolymers at varying concentrations and contacting time (Figure 5). The untreated E. coli

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exhibited a smooth appearance with an intact cell membrane (Figure 5(1A)), without grooves or

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indentations on the cell surface. In the height and section images, the untreated E. coli was elliptical-

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shaped with middle high and end low. After treatment by antimicrobial copolymers with the concentrations

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below MIC and above MIC, E. coli showed different morphologies. The cell profile was yet legible after 11



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exposure to copolymer B4 at 5 ppm (below MIC) for 30 min (Fig. 5(2A)). The cell surface was not

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obviously indented, while only minor leakage occurred around the cell membrane. Section image

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displayed that the height of the bacteria cell dropped from > 200 to < 200 nm, which coincided with the

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OD260 ratio from UV measurement. The final OD260 ratio for B4 increased slightly to 1.09, because the

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intracellular contents were yet mostly maintained in bacteria cells, but only slightly leaked out. The

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bacteria cells were severely damaged and their membranes fully collapsed after exposure to 50 ppm

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copolymer A4 (above MIC) for 8 min (Figure 5(3A)). Only minor debris was found and the bacterial cell

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profile was difficult to identify. The cell membrane was disintegrated and thereby intracellular contents

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were leaked from the cells. Considerable loss of intracellular contents from the cells reduced the bacteria

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height to about 100 nm. The massive cytoplasm remained in bacteria suspension much rose the OD260 to

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1.48 at a high copolymer concentration. AFM showed the antimicrobial mechanism of the copolymers was

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to damage the cell membranes and induce the release of intracellular contents.

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The antibacterial ability of the copolymers was explored via CLSM, which brings more details about the

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bacterial viability and microbial images. All bacteria either living or dead were stained green by FITC and

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photographed after excitation at 488 nm. Only dead bacteria were stained red by PI, indicating the

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antimicrobial ability of the copolymers, and were recorded after excitation at 543 nm. Figure 6 shows the E.

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coli untreated and treated with antimicrobial copolymers at different concentrations and contacting time. In

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the top images, green means both alive and dead E. coli, whereas red stands for dead E. coli in the middle.

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By merging the top and middle images, yellow also represents dead cells on the bottom. For fresh or

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untreated E. coli, most bacteria were active with very few dead. However, after exposure to copolymer A4

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at 50 ppm (>MIC) for 8 min, almost all bacteria were dead. In comparison, only minor bacteria were killed 12


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after 30 min of contact with copolymer B4 at 5 ppm (MIC). AFM and CLSM showed the antimicrobial mechanism of the copolymers was to 13



260

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destroy bacterial cell membranes and stimulate the release of intracellular contents.

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Supporting Information

263

1

264

copolymer B7.

H-NMR and 13C-NMR spectra of GMA-CPF, 1H-NMR spectra of copolymer A6, and 1H-NMR spectra of

265 266

ACKNOWLEDGMENTS

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This work was funded by National Natural Science Foundation of China (51673040), the Natural Science

268

Foundation of Jiangsu Province (BK20171357), the Prospective Joint Research Project of Jiangsu

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Province545 (BY2016076-01), Opening Project of Guangxi Key Laboratory of Clean Pulp & Papermaking

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and Pollution Control (KF201605), NSERC strategic networks Sentinel-Bioactive Paper and Green Wood

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Fiber Products (Canada) and NSF China (No. 51379077).

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Notes

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The authors declare no competing financial interest.

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REFERENCES

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(1) Chang, Y.; McLandsborough, L.; McClements, D. J. Cationic antimicrobial (ε-polylysine)–anionic

277

polysaccharide (pectin) interactions: influence of polymer charge on physical stability and

278

antimicrobial efficacy. J. Agric. Food Chem. 2012, 60(7), 1837-1844.

279 280

(2) Bastarrachea, L. J.; Goddard, J. M. Antimicrobial coatings with dual cationic and N-halamine character: characterization and biocidal efficacy. J. Agric. Food Chem. 2015, 63(16), 4243-4251. 14


Page 15 of 33


281

(3) Ning, Y.; Chen, F.; Xu, X.; Jin, Y.; Wang, Z.; Yang, K.; Jia, Y. Biosynthesis of neokestose laurate

282

catalyzed by Candida antarctica lipase B and its antimicrobial activity against food pathogenic and

283

spoilage bacteria. J. Agric. Food Chem. 2017, 65(50), 11092-11099.

284

(4) Guoruoluo, Y.; Zhou, H.; Zhou, J.; Zhao, H.; Aisa, H. A.; Yao, G. Isolation and characterization of

285

sesquiterpenoids from cassia buds and their antimicrobial activities. J. Agric. Food Chem. 2017, 65(28),

286

5614-5619.

287

(5) Alsager, O. A.; Alnajrani, M. N.; Alhazzaa, O. Decomposition of antibiotics by gamma irradiation:

288

Kinetics, antimicrobial activity, and real application in food matrices. Chem. Eng. J. 2018, 338,

289

548-556.

290

(6) Zhao, R.; Kong, W.; Sun, M.; Yang, Y.; Liu, W.; Lv, M.; Song, S.; Wang, Li.; Song, H.; Hao, R. Highly

291

stable graphene-based nanocomposite (GO−PEI−Ag) with broad-spectrum, long-term antimicrobial

292

activity and antibiofilm effects. ACS Appl. Mater. Interfaces, 2018, 10(21), 17617-17629.

293

(7) Zhou, C.; Chia, G. W. N.; Ho, J. C. S.; Seviour, T.; Sailov, T.; Liedberg, B.; Kjelleberg, S.; Hinks, J.;

294

Bazan, G. C. Informed molecular design of conjugated oligoelectrolytes to increase cell affinity and

295

antimicrobial activity. Angew. Chem. Int. Ed. 2018, 57, 1-5.

296

(8) Venkataraman, S.; Zhang, Y.; Liu, L. H.; Yang, Y. Y. Design, syntheses and evaluation of

297

hemocompatible pegylated-antimicrobial polymers with well-controlled molecular structures.

298

Biomaterials 2010, 31(7), 1751-1756.

299 300 301

(9) Kuroda, K.; DeGrado, W. F. Amphiphilic polymethacrylate derivatives as antimicrobial agents. J. Am. Chem. Soc. 2005, 127(12), 4128-4129. (10) Stratton, T. R.; Rickus, J. L.; Youngblood, J. P. In vitro biocompatibility studies of antibacterial 15



302 303 304 305 306

Page 16 of 33

quaternary polymers. Biomacromolecules 2009, 10(9), 2550-2555. (11) Allison, B. C.; Applegate, B. M.; Youngblood, J. P. Hemocompatibility of hydrophilic antimicrobial copolymers of alkylated 4-vinylpyridine. Biomacromolecules 2007, 8(10), 2995-2999. (12) Oda, Y.; Kanaoka, S.; Sato, T.; Aoshima, S.; Kuroda, K. Block versus random amphiphilic copolymers as antibacterial agents. Biomacromolecules 2011, 12(10), 3581-3591.

307

(13) Sambhy, V.; Peterson, B. R.; Sen, A. Antibacterial and hemolytic activities of pyridinium polymers as

308

a function of the spatial relationship between the positive charge and the pendant alkyl tail. Angew.

309

Chem., Int. Ed. 2008, 120(7), 1270-1274.

310 311

(14) Kuroda, K.; Caputo, G. A.; DeGrado, W. F. The role of hydrophobicity in the antimicrobial and hemolytic activities of polymethacrylate derivatives. Chem.–Eur. J. 2009, 15(5), 1123-1133.

312

(15) Mowery, B. P.; Lindner, A. H.; Weisblum, B.; Stahl, S. S.; Gellman, S. H. Structure−activity

313

relationships among random nylon-3 copolymers that mimic antibacterial host-defense peptides. J. Am.

314

Chem. Soc. 2009, 131(28), 9735-9745.

315 316 317 318

(16) Palermo, E. F.; Kuroda, K. Chemical structure of cationic groups in amphiphilic polymethacrylates modulates the antimicrobial and hemolytic activities. Biomacromolecules 2009, 10(6), 1416-1428. (17) Kenawy, El-Refaie; Worley, S. D.; Broughton, Roy. The chemistry and applications of antimicrobial polymers: A state-of-the-art review. Biomacromolecules 2007, 8(5), 1359-1384.

319

(18) Chen, Chris Zhisheng; Beck-Tan, Nora C.; Dhurjati, Prasad; Dyk, Tina K. van; LaRossa, Robert A.;

320

Cooper, Stuart L. Quaternary ammonium functionalized poly(propylene imine) dendrimers as effective

321

antimicrobials: Structure−activity studies. Biomacromolecules 2000, 1(3), 473-480.

322

(19) Palermo, Edmund F.; Sovadinova, Iva; Kuroda, Kenichi. Structural determinants of antimicrobial 16


Page 17 of 33


323

activity and biocompatibility in membrane-disrupting methacrylamide random copolymers.

324

Biomacromolecules 2009, 10(11), 3098-3107.

325 326

(20) Zhu, W.; Wang, Y.; Sun, S.; Zhang, Q.; Li, X.; Shen, Z. Facile synthesis and characterization of biodegradable antimicrobial poly(ester-carbonate). J. Mater. Chem. 2012, 22, 11785-11791.

327

(21) Dizman, B.; Elasri, M. O.; Mathias, L. J. Synthesis and antibacterial activities of water‐soluble

328

methacrylate polymers containing quaternary ammonium compounds. J. Polym. Sci.: Part A: Polym.

329

Chem. 2006, 44, 5965-5973.

330

(22) Chakrabarty, S.; King, A.; Kurt, P.; Zhang, W.; Ohman, D. E.; Wood, L. F.; Lovelace, C.; Rao, R.;

331

Wynne, K. J. Highly effective, water-soluble, hemocompatible 1,3-propylene oxide-based

332

antimicrobials: Poly[(3,3-quaternary/PEG)-copolyoxetanes]. Biomacromolecules 2011, 12(3), 757-769.

333

(23) Tew, G. N.; Liu, D.; Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll, P. J.; Klein, M. L.; DeGrado, W. F.

334 335 336 337 338 339 340 341 342 343

De novo design of biomimetic antimicrobial polymers. Proc. Natl. Acad. Sci. 2002, 99(8), 5110-5114. (24) Lewis, K.; Klibanov, A. M. Surpassing nature: rational design of sterile-surface materials. Trends Biotechnol. 2005, 23(7), 343-348. (25) Tiller, J. C.; Liao, C.-J.; Lewis, K.; Klibanov, A. M. Designing surfaces that kill bacteria on contact. Proc. Natl. Acad. Sci. 2001, 98(11), 5981-5985. (26) Lin, J.; Tiller, J. C.; Lee, S. B.; Lewis, K.; Klibanov, A. M. Insights into bactericidal action of surface-attached poly(vinyl-N-hexylpyridinium) chains. Biotechnol. Lett. 2002, 24(10), 801-805. (27) Makovitzki, A.; Avrahami, D.; Shai, Y. Ultrashort antibacterial and antifungal lipopeptides. Proc. Natl. Acad. Sci. 2006, 103(43), 15997-16002. (28) Wang, X.; Yang, F.; Yang, W.; Yang, X. A study on the antibacterial activity of one-dimensional ZnO 17



344

Page 18 of 33

nanowire arrays: effects of the orientation and plane surface. Chem. Commun. 2007, (42), 4419-4421.

345

(29) Chen, Y.; Wilbon, P. A.; Chen, Y. P.; Zhou, J.; Nagarkatti, M.; Wang, C.; Chu, F.; Decho, A. W.; Tang,

346

C. Amphipathic antibacterial agents using cationic methacrylic polymers with natural rosin as pendant

347

group. RSC Adv. 2012, 2(27), 10275-10282.

348

(30) Ansari, M. A.; Khan, H. M.; Khan, A. A.; Ahmad, M. K.; Mahdi, A. A.; Pal, R.; Cameotra, S. S.

349

Interaction of silver nanoparticles with Escherichia coli and their cell envelope biomolecules. J. Basic

350

Microbiol. 2014, 54(9), 905–915.

351 352

(31) Fang, F.; Fang, X.; Li, J.; Wei, Z.; Wang, X.; Wang, X. A detailed approach to study the antibacterial mechanisms of nanostructure. Applied Surface Science 2012, 258(10), 4397-4401.

353

(32) Han, Y.; Linton, R. H.; Nielsen, S. S.; Nelson, P. E. Inactivation of Escherichia coli O157: H7 on

354

surface-uninjured and-injured green pepper (Capsicum annuum L.) by chlorine dioxide gas as

355

demonstrated by confocal laser scanning microscopy. Food Microbiology 2000, 17(6), 643-655.

356

(33) Drummond, D. R.; Carter, N.; Cross, R. A. Multiphoton versus confocal high resolution z-sectioning

357

of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal

358

plane can be compensated using a Pockels cell and dual widefield detectors. Journal of microscopy

359

2002, 206(2), 161-169.

360

(34) Li, A.; Lee, P. Y.; Ho, B.; Ding, J. L.; Lim, C. T. Atomic force microscopy study of the antimicrobial

361

action

of Sushi

peptides

on

Gram

negative bacteria. Biochimica

362

(BBA)-Biomembranes 2007, 1768(3), 411-418.

et

Biophysica

Acta

363

(35) Rossetto, G.; Bergese, P.; Colombi, P.; Depero, L. E.; Giuliani, A.; Nicoletto, S. F.; Pirri, G. Atomic

364

force microscopy evaluation of the effects of a novel antimicrobial multimeric peptide on 18


Page 19 of 33


365

Pseudomonas aeruginosa. Nanomedicine: Nanotechnology, Biology and Medicine 2007, 3(3), 198-207.

366

(36) Meincken, M.; Holroyd, D. L.; Rautenbach, M. Atomic force microscopy study of the effect of

367

antimicrobial peptides on the cell envelope of Escherichia coli. Antimicrobial agents and

368

chemotherapy 2005, 49(10), 4085-4092.

369 370

(37) Chen, C. Z.; Cooper, S. L. Interactions between dendrimer biocides and bacterial membranes. Biomaterials 2002, 23(16), 3359-3368.

371

(38) Oonmetta-Aree, J.; Suzuki, T.; Gasaluck, P.; Eumkeb, G. Antimicrobial properties and action of

372

galangal (Alpinia galanga Linn.) on Staphylococcus aureus. LWT-Food Science and Technology 2006,

373

39(10), 1214-1220.

374 375

(39) Liu, H.; Du, Y.; Wang, X.; Sun, L. Chitosan kills bacteria through cell membrane damage. International journal of food microbiology 2004, 95(2), 147-155.

376

(40) He, M.; Xiao, H. N.; Zhou, Y. M.; Lu, P. Synthesis, characterization and antimicrobial activities of

377

water-soluble amphiphilic copolymers containing ciprofloxacin and quaternary ammonium salts. J.

378

Mater. Chem. B 2015, 3, 3704-3713.

379 380

(41) He, M.; Zhou, Y. M.; Xiao, H. N.; Lu, P. Amphiphilic cationic copolymers with ciprofloxacin: preparation and antimicrobial activities. New J. Chem. 2015, 40(2), 1354-1364.

381

(42) Xue, Y.; Guan, Y.; Zheng, A.; Wang, H.; Xiao, H. Synthesis and characterization of ciprofloxacin

382

pendant antibacterial cationic polymers. Journal of Biomaterials Science, Polymer Edition 2012, 23(8),

383

1115-1128.

384

(43) Ortega, P.; Cobaleda, B. M.; Hernández-Ros, J. M.; Fuentes-Paniagua, E.; Sánchez-Nieves, J.;

385

Tarazona, M. P.; Copa-Patiño, J. L.; Soliveri, J.; Javier de la Mata, Fco.; Gómez, R. Hyperbranched 19



Page 20 of 33

386

polymers versus dendrimers containing a carbosilane framework and terminal ammonium groups as

387

antimicrobial agents. Org. Biomol. Chem. 2011, 9, 5238-5248.

388 389 390 391

(44) Mitra, R. N.; Shome, A.; Paul, P.; Kumar Das, P. Antimicrobial activity, biocompatibility and hydrogelation ability of dipeptide-based amphiphiles. Org. Biomol. Chem. 2009, 7, 94-102. (45) Dizman, B.; Elasri, M. O.; Mathias, L. J. Synthesis, characterization, and antibacterial activities of novel methacrylate polymers containing norfloxacin. Biomacromolecules 2005, 6(1), 514-520.

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FIGURE CAPTIONS: Figure 1. Synthesis of amphiphilic copolymers containing CPF, alkyl group and different amine functional groups in the side chains. Figure 2. Thermogravimetric analysis of antimicrobial copolymers and CPF. Figure 3. Antimicrobial activities of amphiphilic copolymers containing CPF, alkyl group and primary or quaternary amine groups in the side chains against E. coli. Figure 4. Dynamic UV absorption of E. coli suspension treated with antimicrobial copolymers at different concentrations. Figure 5. Morphology of E. coli treated with antimicrobial copolymers with different concentration. Figure 6. Confocal images of E. coli untreated and treated by antimicrobial copolymers. Note: green dots on the top are alive and dead E. coli; red dots in the middle are dead E. coli; yellow dots on the bottom are dead E. coli and green dots are alive ones. Figure 7. CLSM images of E. coli cells untreated and treated by antimicrobial copolymers

21



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Table 1. Reaction parameters for the polymerization of AEMAH(M1), GMA-CPF(M2) and BA(M3) and copolymers compositions Monomer feed compositions M1(mol%)

a

M2(mol%)

M3(mol%)

Copolymers compositions M1(mol%)a

M2(mol%) a

M3(mol%) a

A1

85

5

10

87.9

2.3

9.8

A2

81

5

14

83.0

2.3

14.7

A3

76

5

19

76.8

2.2

21.0

A4

71

5

24

70.4

2.2

27.4

A5

66

5

29

65.0

2.2

32.8

A6

62

5

33

62.0

2.3

35.7

A7

48

10

42

47.3

4.4

48.3

Mole percentages of M1, M2 and M3 in the copolymers were calculated from analysis of the peak

integration of the 1H NMR spectra, as described in Supporting Information (the same below).

22


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Table 2. Reaction parameters for the polymerization of DMAEAMC(M1), GMA-CPF(M2) and BA(M3) and copolymers compositions Monomer feed compositions M1(mol%)

M2(mol%)

Copolymers compositions

M3(mol%)

M1(mol%)

M2(mol%)

M3(mol%)a

B1

75

5

20

76.3

2.2

21.5

B2

70

5

25

71.3

2.3

26.4

B3

65

5

30

65.5

2.3

32.2

B4

62

5

33

62.0

2.2

35.8

B5

58

5

37

58.8

2.2

39.0

B6

53

5

42

53.0

2.2

44.8

B7

48

5

47

47.8

2.2

50.0

B8

75

10

15

75.6

4.4

20.0

23



Page 24 of 33

Table 3. Minimum inhibition concentrations of copolymers with various monomer ratios against E. coli Copolymers compositions

AEMAH content (mol%) GMA-CPF content (mol%) BA content (mol%)

MIC (ppm)

A1

87.9

2.3

9.8

31.2

A2

83.0

2.3

14.7

12.0

A3

76.8

2.2

21.0

4.0

A4

70.4

2.2

27.4

2.0

A5

65.0

2.2

32.8

2.0

A6

62.0

2.3

35.7

2.0

A7

47.3

4.4

48.3

10.0

24


Page 25 of 33


Table 4. Minimum inhibition concentrations of copolymers with various monomer ratios against E. coli Copolymers compositions DMAEAMC content (mol%)

GMA-CPF content (mol%) BA content (mol%)

MIC (ppm)

B1

76.3

2.2

21.5

62.5

B2

71.3

2.3

26.4

31.2

B3

65.5

2.3

32.2

15.6

B4

62.0

2.2

35.8

10.0

B5

58.8

2.2

39.0

7.8

B6

53.0

2.2

44.8

10.0

B7

47.8

2.2

50.0

12.0

B8

75.6

4.4

20.0

31.2

CPF

-

-

-

7.0

25



Figure 1.

26


Page 26 of 33

Page 27 of 33


100

Copolymer A4 Copolymer B4 CPF

Weight (%)

80 60 40 20 0

0

100 200 300 400 500 600 700 800 900

Temperature (oC)

Figure 2.

27



60

AEMAH/BA/GMA-CPF DMAEAMC/BA/GMA-CPF

MIC (ppm)

50 40 30 20 10 0

10

20

30

40

BA content (%)

Figure 3.

28


50

Page 28 of 33

Page 29 of 33


A

1.56

A4(MIC2.0) B4(MIC10.0) A1(MIC31.2)

O.D. ratio (260 nm)

1.52 1.48 1.44 1.40 1.36 1.32 1.28 1.24 1.20 0

10

20

30

40

50

60

70

80

Time (min)

(a) 50ppm copolymer solution B

1.50 A4(MIC2.0) B4(MIC10.0) A1(MIC31.2)

O.D. ratio (260 nm)

1.45 1.40 1.35 1.30 1.25 1.20 1.15 1.10 1.05 1.00 0

10

20

30

40

50

60

70

Time (min)

(b) 5ppm copolymer solution

Figure 4.

29


80


A

B

C

（1）fresh E. Coli

(2) Copolymer B4 (5ppm, 30min) (3) Copolymer A4 (50ppm, 8min)

Figure 5.

30


Page 30 of 33

Page 31 of 33


(1) fresh E. coli

(2) Copolymer B4 (5ppm 30min)

Figure 6.

31


(3) Copolymer A4 (50ppm 8min)


(1) fresh E. coli

(2) Copolymer B4 (5ppm 30min)

Figure 7.

32


(3) Copolymer A4 (50ppm 8min)

Page 32 of 33

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Table of Contents Graphic

33


Synthesis of Amphiphilic Copolymers Containing ... - ACS Publications

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