Outstanding Antibiofilm Features of Quanta-CuO Film on Glass Surface

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Outstanding Antibiofilm Features of Quanta-CuO Film on Glass Surface Nirmalya Tripathy, Rafiq Ahmad, Seung Hyuck Bang, Gilson Khang, Jiho Min, and Yoon-Bong Hahn ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04494 • Publication Date (Web): 01 Jun 2016 Downloaded from http://pubs.acs.org on June 7, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Outstanding Antibiofilm Features of Quanta-CuO Film on Glass Surface Nirmalya Tripathy*†, Rafiq Ahmad‡, Seung Hyuck Bang§, Gilson Khang†, Jiho Min§, and YoonBong Hahn*‡ † Department of BIN Fusion Technology, Department of PolymerNano Science & Polymer BIN Research Center, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do, 54896 Republic of Korea ‡ School of Semiconductor and Chemical Engineering, Nanomaterials Processing Research, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do, 54896 Republic of Korea. § Department of Bioprocess Engineering, Chonbuk National University, 567 Baekje-daero, Deokjin-gu, Jeonju-si, Jeollabuk-do, 54896 Republic of Korea *Corresponding author: [email protected] (N. Tripathy), [email protected] (Y. B. Hahn)

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 37

ABSTRACT

Intelligently designed surface nano-architecture provides defined control over the behavior of cells and biomolecules at the solid-liquid interface. In this study, CuO quantum dots (quantaCuO; ~3-5nm) were synthesized by a simple, low-temperature solution process and further formulated as paint to construct quanta-CuO thin film on glass. Surface morphological characterizations of the as-coated glass surface reveal a uniform film thickness (~120±10nm) with homogeneous distribution of quanta-CuO. Antibiofilm assay showed very high contact bacteria-killing capacity of as-coated quanta-CuO glass surfaces towards Staphylococcus aureus and Escherichia coli. This efficient antibacterial/antibiofilm activity was ascribed to the intracellular ROS generated by the quanta-CuO attached to the bacterial cells, which leads to an oxidative assault and finally results in bacterial cell death. Although, there is a significant debate going on the CuO nanostructure’s antibacterial mode of action, however we propose both contact killing and/or copper ion release killing mechanisms for the antibiofilm activity of quanta-CuO paint. Moreover, synergism of quanta-CuO with conventional antibiotics was also found to further enhance the antibacterial efficacy of commonly used antibiotics. Collectively, this stateof-the-art design of quanta-CuO coated glass can be envisioned as promising candidates for various biomedical and environmental device coatings.

KEYWORDS: quanta-CuO; film; glass; antibacterial; antibiofilm; antibiotics


2

Page 3 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


INTRODUCTION Bacterial colonization and biofilm development triggered by biomedical implants, in-dwelling devices, industrial pipes and instruments are still remains major issues for outbreak of several infectious diseases as well as hospital-acquired infection.1 In general, typical biofilm mode of growth is often featured with inherent resistance to antimicrobial treatments and immune response killing. As a result, conventional antibiotic treatments against device-associated biofilm microorganisms usually ineffective without elimination of infected implant.2,3 Approximately 1.7 million hospitalized patients were affected by nosocomial infections in 2002, of which almost half of all nosocomial infections were device-related, with the greatest percentage of cases consisting of urinary tract and central venous catheterization related infections.4,5 Addressing such undesirable situations, various antimicrobial coatings/films strategies were fabricated such as antibiotics,6,7 quaternary ammonium salts,8 cationic peptides,9 etc. However, syntheses of antibiofilm surfaces are complex and expensive, and often tend to lose its effectiveness owing to leaching or depletion of antimicrobial agents.7 Thus these coating or deposition strategies for large-area antimicrobial coatings were mainly associated with shortcomings related to its stability, fabrication protocols demanding sophisticated instrumental setup and its associated high cost.10,11 Addressing such undesirable situations, intelligently designed nanostructured surfaces that provide effective control over biomolecules/cells/tissues deposition and growth were highly demanded. Regarding this context, metallic and non-metallic nanomaterials/nanostructures (mostly nanoparticle or quantum dots) have shown great promise in the biomedical domain as potent antimicrobial entities because of easy synthesis, high yield with large surface area to volume ratio and versatile physiochemical features.12-15 Importantly, antimicrobial nanomaterials have


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 37

been reported to tackle multiple biological pathways dominant in broad range of microbes and thus many concurrent mutations would require for developing resistance against these nanomaterials antimicrobial activities. Compared with conventional antibiotics synthesis, nanomaterials antimicrobial can be easily and cost-effectively synthesized with long-term storage stability and prolonged shelf life. Some nanomaterials can also withstand harsh conditions, for example high temperature sterilization, however under which conventional antibiotics usually gets inactivated.16 Although silver nanoparticles were well-established antibacterial agents since ancient time and known as most effective among metallic nanomaterials, however its antibacterial activity varies depending on the microbial species.17-29 Recently, inorganic metal oxides (such as ZnO, TiO2, MgO and Al2O3) were garnered increasingly attention from researchers as an efficient material for tailoring antimicrobial coatings and thus actively replacing the frequently used silver because of its varied toxic effects on human health and environment.20,21 Copper oxide (CuO), among the large metal oxides family, has emerged as an important class of materials with varied applications for conductive inks, catalysts and antimicrobial agents.22,23 It was reported that copper nanoparticles have greater affinity to carboxyl groups and amines at a high density on Bacillus subtilis outer surfaces compared to silver nanoparticles, thus demonstrates superior antibacterial activity.24,25 CuO is quite cheaper than silver, easily miscible with polymers and relatively stable with long shelf life in terms of both chemically and physically.26 Moreover, copper is regarded as one of small group of metallic elements which are essential for human health.27 Additionally, human tissues (skin or others) demonstrates enhanced resistance to copper as compared to microorganisms.28 Addressing the antimicrobial mechanism of CuO, Li et al. attributed the antibacterial action of CuO nanoparticles to its relatively high solubility or dissolution in


4

Page 5 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


biological medium and the impacts of released copper ions leading to ROS production.29 Thus there is a general consensus that bacterial toxicity of CuO nanomaterials is mostly due to oxidative pathways, however there is still ongoing debate persists about the relative importance of the particle surface and released ions in primary ROS generation.30 Despite of its known beneficial applications, none of the studies have explored the possibility of using CuO nanomaterials as robust coating material to inhibit biofilm formation on glass surfaces. Inspired by those intriguing features of CuO nanomaterials, we report herein a naive antibiofilm coating strategy by employing well-designed, uniformly dispersed, low-temperature synthesized CuO quantum dots (quanta-CuO). The strategy utilizes the formulation of quantaCuO paint with ethanol, isopropyl alcohol, polydopamine and poly(ethylene glycol) and then spin casted onto the glass surfaces followed by antibiofilm/antibacterial study. The resulting quanta-CuO coated glass surfaces not only resisted bacterial cells interaction and attachment but also found to kill two clinically relevant Gram-positive and Gram-negative bacteria i.e. Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli), respectively. Furthermore, a short pre-exposure to the quanta-CuO coated glass surfaces also showed enhanced bacterial susceptibility towards commonly used conventional antibiotics. In addition, the involved mechanism for antibacterial/antibiofilm activity was highlighted in detail.

EXPERIMENTAL SECTION Materials: Copper acetate dihydrate, acetic acid, sodium hydroxide (NaOH), ethanol, phosphatebuffered saline (PBS, pH 7.14), 5,5-dimethyl-1-pyrroline- N-oxide (DMPO), CM-H2DCFDA [5(and 6)chloromethyl-2,7-dichlorodihydrofluorescein diacetate, acetyl ester], poly(ethylene


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 37

glycol) and hydroxyethyl cellulose (HEC) were purchased from Sigma Aldrich (St Louise, MO, USA). The kits used for biofilm imaging was Live/Dead BacLight kit from Invitrogen (Carlsbad, CA, USA). Synthesis of quanta-CuO: The typical experimental procedure for quanta-CuO synthesis was as follows: copper acetate dihydrate (0.02 M) and acetic acid (1 mL) was dissolved in 100 mL ethanol in a round bottom flask under vigorous stirring, while keeping the solution temperature at 75 oC. Next, the solution pH = 5.5 was maintained by adding NaOH (0.6 gm). Once the solution turns dark brown in color, the reaction was continued for 20 min followed by repeated washing with ethanol by centrifugation to remove the by-products (sodium acetate) and then redispersed in ethanol to form a stable quanta-CuO suspension. Quanta-CuO paint formulation and thin film fabrication on glass: Firstly, the quanta-CuO paint was formulated using 30 wt% quanta-CuO mixed with ethanol, isopropyl alcohol, and poly(ethylene glycol) in the ratio of 40:10:10 by volume %, and stirred for 1 h. Then 10 wt % polydopamine (a well-known adhesive and anti-bacterial agent) was added to the above solution with continuous stirring for 24 h and filtered using 0.45 µm polypropylene whatman paper. Next, a viscosifying agent (2 wt% HEC in ethanol) was added to the above solution to achieve 3:100 ratio (by weight) of HEC:CuO. Prior to the thin film fabrication, glass substrates were cleaned with 2-propanol, sterile water, acetone and dried. Then the quanta-CuO thin films were deposited on glass substrates by spin casting the above solution at 1000 rpm for 30s and finally coated samples were dried on hot plate at 100 °C for 10 min. The morphological and structural characterizations of quanta-CuO were investigated by transmission electron microscopy (TEM) and high-resolution TEM (HRTEM) equipped with


6

Page 7 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


selected area electron diffraction (SAED) pattern. The crystallinity and optical properties of quanta-CuO was examined by X-ray diffraction (XRD, Rigaku) with Cu Kα radiation (λ = 1.54178 Å) in the range of 25-70° at 40 kV and Raman-scattering measurements with Ar+ (513.4 nm) as the exciton source, respectively. The bonding characteristics of samples were studied by X-ray and ultraviolet photoelectron spectroscopy (XPS, UPS) spectra with a Thermo K-alpha ESKA System with a monochromatic Al-kα source and a charge neutralizer. The thickness of quanta-CuO film on glass surface was evaluated by field emission scanning electron microscopy (FESEM, Hitachi 4800 JSM) images. Furthermore, the hydrophilicity of samples was measured by analyzing water droplet angle formed between the liquid/solid interfaces on the films using water contact goniometer (TantecTM, CAM-PLUS Micro, USA) (n = 10). The depth and adhesion profile of quanta-CuO coatings were characterized by rutherford backscattering spectroscopy (RBS) microbeam analysis, carried with a 3.0 MeV He+ beam produced by Tandetron 1.7 MV accelerator of high voltage engineering. The analysis conditions were: current density, 1 nA; spatial resolution, about 2 mm; mapping area of 500 x 500 µm2. Antibiofilm tests: In this study, we have chosen bacterial strains from two different species as representatives of various bacteria types i.e. Gram positive (S. aureus) and Gram negative (E. coli) owing to its potent capability to form biofilm. Preliminarily, the antibacterial efficacy of assynthesized quanta-CuO was evaluated on solid agar plates at different concentrations ranging from 50-250 µg/mL. The bacteria strains (S. aureus and E. coli) were cultured in 100 mL nutrient broth medium and maintained at 37 oC shaking incubator up to the optical density (OD600) reached 0.7-0.8. Then the total number of cells was further diluted to 106 cells/mL in sterilized distilled water and the cell cultures (100 µL) with quanta-CuO in PBS (900 µL) were spread over solid agar plates followed by incubation for 12 h at 37 oC. The antibacterial efficiency of the


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 37

samples was measured by calculating colony-forming units (CFU) per plate (estimated as 200 CFU/plate). Thereafter, the antibiofilm performance of quanta-CuO coated glass samples were studied using two bacterial biofilm continuous flow cell models. These models were inoculated with 0.3 OD600 (~ corresponds to 3 x 108 CFU/mL) dilution of S. aureus and E. coli culture overnight and the flow was initiated after 1 h incubation at 37 °C with 10 mL h-1 flow rate, and 1% TSB-Glu or 1% TSB (diluted in double distilled water) was employed as growth medium for S. aureus and E. coli, respectively. After completion of incubation period, the samples were removed from experimental flow cells, washed with distilled water to remove unattached cells and subjected to microscopic analysis of the biofilm formation. For CLSM (confocal laser scanning microscopy, LSM 510 META, Carl Zeiss), samples were stained using Live/Dead BacLight reagent (Invitrogen, Carlsbad, CA, USA) according to manufacturer’s instructions. The viable bacteria with intact cell membranes were stained in green dye (fluorescein isothiocyanate; FITC), whereas dead bacteria cells with damaged membranes stained in red dye (propidium iodide; PI) were observed under CLSM at λexcitation = 488 nm and λemission = 515 nm. All the captured images were analyzed with image analysis software (LCS image browser). Negative controls were prepared without quantum dots. All experiments were performed in triplicates. ROS detection: The electron spin resonance (ESR) spectroscopy (Bruker EMX spectrometer; Bruker Instruments, Billerica, MA) measurements were conducted using ESR spin trapping technique by employing a spin trap DMPO (0.02 M). The spin trap was added to the aqueous medium containing quanta-CuO coated glass samples (1 cm x 1 cm); and was drawn using a syringe into the gas permeable Teflon capillary (Zeus Industries, Raritan, NJ); inserted into the narrow quartz tube with both opened ends and then placed in ESR cavity. Next, the obtained


8

Page 9 of 37

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


spectra were recorded and processes using Bruker WIN-ESR software version 2.11. The used ESR measurement conditions were as follows: frequency -9.74 GHz, microwave power -20 mW, scan width -65 G, receiver gain -2 x 105, resolution -1024; conversion time -82 ms, sweep time 84 s, time constant -655 ms, number of scans -2, and modulation frequency -100 kHz. Furthermore, the rate of intracellular ROS generated in the bacteria was also evaluated by monitoring their reaction with fluorescent dye i.e. CM-H2DCFDA. Cells were pre-incubated with CM-H2DCFDA (10 µM in PBS) for 15 min, which allows the dye to permeate bacterial cells. This dye can freely enter bacterial cell membrane and turns fluorescent upon cleaved by intracellular ROS. The cells were washed twice with PBS prior to CLSM. All the samples were recorded at an excitation = 485 nm and an emission = 535 nm. Untreated and DCFH-DA incubated cells treated with 100 µM H2O2 for 30 min were denoted as negative and positive controls, respectively. Combined effect of quanta-CuO coating and antibiotics on glass: First, the minimal inhibitory concentrations of the tested antibiotics were detected. The antibiotics employed herein were Ampicillin (AM), Nalidixic acid (NA), Chloramphenicol (CP) for both stains; Tobramycin (TO) for S. aureus and Erythromycin (ER) for E. coli. Test tubes containing different antibiotics concentrations were inoculated with bacterial suspension (100 µL; ~105 CFUmL-1) for 24 h at 37 o

C and the growth were assessed by measuring OD at 660nm. Furthermore, two types of agar

media were prepared: (i) without antibiotics termed as non-selective media; (ii) with only one antibiotic (at half of its MIC) termed as selective media. The experimental protocol was as follows: tested bacteria were cultured on nutrient agar overnight; transferred into a nutrient broth (0.1 OD600) and grown at 37 oC. Once 0.3 OD was obtained, the cultures were centrifuged and saline washed to obtain a final bacterial concentration of ~105 CFUmL-1. An aliquot (4.5mL) of


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 37

saline solution containing coated and uncoated glass (1cm x 1cm) was then added to cells (500 µL) and incubated for 15 min. Samples (each 100 mL) were diluted in saline solution and plated onto non-selective, selective and nutrient agar plates. Next, these plates were cultured for 24 h at 37 oC followed by viable cells counting and subsequently comparing the number of colonies on both media. Negative control plates were performed with an uncoated glass. Statistical analysis: All the results were presented as means ± SD (Standard Deviation) of experiments performed three times independently. Statistical significance was studied using ANOVA (one-way analysis of variance) followed by Dunnett's multiple comparison test with the assistance of Graph pad software. *P values

Outstanding Antibiofilm Features of Quanta-CuO Film on Glass Surface

Recommend Documents