Fabrication of Antimicrobial Poly(propylene carbonate) Film by Plasma

Oct 19, 2017 - ⊥School of Pharmacy and Medical Sciences and ∥Adelaide Biofilm Test Facility, Sansom Institute for Health Research, University of S...
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Fabrication of Antimicrobial Poly(Propylene Carbonate) Film by Plasma Surface Modification Bahareh Bahramian, Wojciech Chrzanowski, Alexey Kondyurin, Nicky Thomas, and Fariba Dehghani Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.7b01185 • Publication Date (Web): 19 Oct 2017 Downloaded from http://pubs.acs.org on October 27, 2017

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Industrial & Engineering Chemistry Research 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.

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Industrial & Engineering Chemistry Research

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Fabrication

of

Antimicrobial

Poly(Propylene

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Carbonate) Film by Plasma Surface Modification

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Bahareh Bahramiana, Wojciech Chrzanowskib, Alexey Kondyurinc, Nicky Thomasd,e, Fariba

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Dehghania,*

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The University of Sydney, aSchool of Chemical & Biomolecular Engineering, bFaculty of

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Pharmacy, cSchool of Physics, Sydney, NSW 2006, Australia

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d

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Australia

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e

University of South Australia, School of Pharmacy and Medical Sciences, Adelaide, SA 5000,

University of South Australia, Adelaide Biofilm Test Facility, Sansom Institute for Health

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Research, Adelaide, SA 5000, Australia

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E-mail: [email protected];

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Keyword: poly(propylene carbonate), antimicrobial polymers, plasma surface modification,

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thymol

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ABSTRACT

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Plasma coating was used as a green process for immobilization of thymol, a natural antimicrobial

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compound, on the surface of Poly(propylene carbonate) (PPC). PPC is a partially renewable

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polymer that is synthesized from CO2 and degrades into benign products including water and

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CO2 and has superior properties for broad ranges of applications. The results of FTIR and water

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contact angle analyses demonstrated that plasma treatment was an efficient for functionalization

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of PPC and immobilizing thymol. Plasma treatment of the PPC surface reduced thymol elution in

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90% alchohol from 60% to 20% (P99.5% purity), peptone,

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yeast extract, D-(+)-Glucose, agar and nutrient agar powder were purchased from Sigma-

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Aldrich. Tryptic Soy Broth (TSB) was bought from BactoTM. Ethanol and glycerol were supplied

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from Merck, and antibiotic-antimycotic (100x) (Anti-Anti) from Invitrogen™. All materials

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were used as received. Escherichia coli (ATCC 25922), and Bacillus subtilis 168 were supplied

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from American Type Culture Collection.

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2.1. Preparation of Antimicrobial PPC Films

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Antimicrobial PPC films were fabricated using both physical coating and plasma treatment

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followed by thymol coating. According to our preliminary study for physical coating, the

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optimum concentration of thymol on PPC films was varied between 1.25 and 2.5 mg/cm2. PPC

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films were prepared by casting technique using acetone as a solvent.

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Thymol physical coating. After the preparation of pure PPC films, a solution of thymol in ethanol

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was dispersed on the casted PPC films. The samples were then dried under ambient conditions to

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remove solvent residue.

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Plasma treatment followed by thymol coating. Plasma was generated in the chamber of PDC-002

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HARRIK PLASMA using room air as plasma gas. After placing the PPC film into the chamber,

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it was modified under high vacuum. Then, the plasma power was turned on followed by feeding

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air into the chamber. Samples were exposed to plasma for 5, 10, or 15 min, and various power

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levels (low–10, medium–20 and high–30W) were applied for PPC samples to examine the effect

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of plasma power on the PPC surface activation. Immediately after activation by plasma, PPC

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films were coated with thymol solution in ethanol and dried at ambient conditions to remove

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solvent residue. The surface of PPC samples were characterized immediately after plasma

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treatment and after coating with thymol to examine the effect of plasma power, time of exposure,

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and thymol concentration on the PPC surface properties.

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2.2. Attenuated Total Reflectance-Fourier Transform Infrared Spectroscopy (ATR-FTIR)

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The effect of plasma activation and formation of antimicrobial layer on the surface of PPC films

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was examined with Attenuated Total reflection Fourier Transform Infrared Spectroscopy (ATR-

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FTIR). The ATR-FTIR spectra of PPC films were collected at a resolution of 1 cm-1 from 64

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scans with an (FTIR) spectrometer (Thermo Scientific Nicolet 6700) fitted with an attenuated-

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total-reflection trapezium germanium crystal over the range of 600-4000 cm-1. To characterize

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the effect of surface treatments, subtracted spectra form the samples before and after plasma

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exposure and thymol coating were analyzed using Grams and Resolution Pro software. Coated

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samples were washed with ethanol 3 times (for 2 min) prior to FTIR analysis to confirm the

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attachment and stability of thymol on the surface of films. The FTIR chamber was dried using

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silica-gel for 6 hours prior to the sample collection to eliminate the effect of humidity on the

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sample collection. The spectra were normalized against untreated PPC and water vapor spectrum

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was subtracted.

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2.3. Water Contact Angle Measurements

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The effect of plasma treatment and thymol coating on the hydrophobicity of PPC surface was

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measured using water contact angle technique. The drop shape analysis with drop shape

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tensiometer (KRUSS-DSA25) was used for this study. In each test 0.8 µl of water was dropped

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on the film surface and right and left contact angles were measured.

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2.4.

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The tensile strength was measured using a universal testing instrument (Instron 5543) equipped

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with a 100 N load-cell. Testing was performed according to ASTM standards using dumbbell

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shaped samples cut from polymer films.

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2.5. Thymol Elution Assay

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Release profile of thymol into aqueous media with 10% and 90% (v%) ethanol was measured to

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assess the stability and attachment of thymol on the surface of PPC films 38. Thymol-coated PPC

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films with the surface area of 10 cm2 were immersed in the 25 ml of the media and stored at

Mechanical Properties

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ambient condition. Samples were collected from the media as a function of time (up to 8 days)

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and analyzed using Agilent Cary 60 UV-Vis Spectrophotometer at the wavelength of 274 nm to

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determine the concentration of released thymol. At the first stage, thymol containing media

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(concentrations 5-100 ppm) were analyzed to plot a calibration curve (R2>0.99). Samples were

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diluted prior to analysis ensure thymol absorbance within the range of calibration curve.

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2.6. Antimicrobial Activity

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High CFU culture media of E. coli (17.8 log CFU/ml) and B. subtilis (10 log CFU/ml) were used

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in this study to demonstrate application of PPC film for packaging food products that commonly

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contained lower microbial level

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more than one month prior to the antimicrobial test to assess the effect of storage at ambient

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conditions on the stability of antimicrobial properties. Two different methods were used to

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examine the antimicrobial activity of PPC/Thymol films. In the first method, known as agar disc

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diffusion, the test culture E. coli and B. subtilis were grown in a previously prepared media

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(containing 4 g peptone, 2 g glycerol, 10 g yeast in 200 mL of MQ water for E. coli, and 27.5 g

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of Tryptic Soy Broth and 2.5 g of glucose per 1000 mL of MQ water for B. subtilis) at 37°C for

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E. coli and 30°C for B. subtilis overnight 40. The Colony Formation Unit (CFU) was counted by

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serial dilutions following by inoculation on agar plates and incubating at 37°C for 16-18 hours.

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Subsequently, 3-4 well-separated colonies were taken from the agar plates and suspended in 3 ml

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media. The suspension again was incubated at 37°C for another 8 hours. After that, disk shaped

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samples with diameter of 8 mm were placed on agar plates which previously spread with 10 µl of

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the bacteria culture. The plates then incubated at 37°C overnight and visually examined for

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inhibition zones around the films, and the diameter of this zone was measured using calipers.

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. Thymol immobilized films were kept at room condition for

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Pure PPC films were applied as negative control and one drop of Anti-Anti on filter paper with

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same size was used as positive control.

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In the second method, PPC/thymol samples were soaked in the separated suspensions of the

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cultured E. coli and B. subtilis and incubated at 37°C for different time intervals. At each time

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point, suspensions were inoculated on agar plates and their Colony Forming Units were

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calculated after 16-18 hour incubation at 37°C.

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2.7. Biofilm Formation Study

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An overnight culture of E. coli and B. subtilis in tryptic soy broth was adjusted to an optical

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density (OD600) of 0.25 in sterile saline (0.9%), corresponding to a cell density of approximately

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3x108 CFU/mL. The suspension was diluted (1/15) with sterile TSB and was used as the

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inoculum for biofilm formation

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sterilized by UV light for 15 minutes before adding 3 mL of the inoculum. Biofilms were

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allowed to grow statically for 24 hours at 37˚C (E. coli) or 30˚C (B. subtilis). The medium was

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then removed from the dishes and the films were carefully washed twice with sterile saline to

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remove adherent planktonic cells. Biofilms were collected from the PPC films using a cell

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scraper and sterile saline (3 × 2.5 mL). The cell suspension was serial-diluted (1/10) in sterile

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saline and plated on TSA plates. Following incubation for 18 hours the number of CFU were

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enumerated.

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2.8. Statistical Analysis

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Data was reported as mean ± STD. One way analysis of variance (ANOVA) was performed

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using Excel for single comparisons. Statistical significance was accepted at p