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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
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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
