Grafted Polymer Coatings Enhance Fouling Inhibition by an

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Biological and Environmental Phenomena at the Interface

Grafted Polymer Coatings Enhance Fouling Inhibition by an Antimicrobial Peptide on Reverse Osmosis Membranes Nitzan Shtreimer-Kandiyote, Tehila Avisdris, Christopher J. Arnusch, and Roni Kasher Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03851 • Publication Date (Web): 21 Dec 2018 Downloaded from http://pubs.acs.org on December 23, 2018

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Grafted Polymer Coatings Enhance Fouling Inhibition by an Antimicrobial Peptide on Reverse Osmosis Membranes Nitzan Shtreimer Kandiyote, Tehila Avisdris, Christopher J. Arnusch* and Roni Kasher*

Department of Desalination and Water Treatment, Zuckerberg Institute for Water Research, The Jacob Blaustein Institutes for Desert Research, Ben-Gurion University of the Negev, Sede-Boqer Campus, 84990, Israel

*Corresponding authors: Tel 972-8-6563532, Fax 972-8-656-3501, Email: [email protected] (CJA); Tel 972-8-6563531, Fax 972-8-656-3501, Email: [email protected] (RK).

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Abstract. Bacterial biofilms that are formed on surfaces are highly detrimental to many areas in industry and medicine. Seawater desalination by reverse osmosis (RO) suffers from biofilm growth on the membranes (biofouling), which limits its widespread use because biofouling decreases water permeance and necessitates module cleaning and replacement, leading to increased economic and environmental costs. Antimicrobial peptides (AMPs) bound covalently to RO membranes inhibit biofilm growth and might delay membrane biofouling. Here we examined how various hydrophilic membrane coatings composed of zwitterionic, neutral, positively charged, and poly(ethylene glycol) (PEG) grafted polymers affected the biocidal activity and the biofilm inhibition of a covalently bonded AMP on RO membranes. The AMP magainin-2 was linked by the copper-catalyzed azide-alkyne cycloaddition reaction to series of RO membranes that were grafted with different methacrylate polymers. Surface characterization by infrared spectroscopy, X-ray photoelectron spectroscopy, and water drop contact angle gave evidence of successful RO modifications, and zeta-potential analysis reflected the increase in surface charge due to the linked positively charged peptide. All AMP-modified membranes inhibited Pseudomonas aeruginosa growth compared with unmodified membranes, and the grafted methacrylic polymers did not significantly interfere with the peptide activity. On the other hand, membranes coated with zwitterionic and other acrylate polymers including AMP attachment inhibited biofilm growth more than either the AMP or the polymer coating alone. This enhancement led to ~ 20% less biofilm biovolume on the membrane surfaces. The combination of antimicrobial coatings with polymer coatings known to resist fouling might aid future designs of surface coatings susceptible to biofilm growth. Keywords: Antimicrobial peptide; graft polymerization; magainin-2; reverse osmosis membranes; biofouling.


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1. Introduction Bacterial adhesion onto surfaces is a significant challenge in diverse applications and leads to biofilm coated surfaces, which must be vigorously cleaned or replaced.1-3 Ubiquitous in nature, the biofilm is part of microbe’s natural life cycle, but can be dangerous if present inside the human body or on medical devices. Biofilms are also detrimental to many industrial and environmental systems that are exposed to the marine environment.3-5 Consequently, antibacterial surfaces have received much research interest, a strategy that might interfere with bacteria adhesion.6 Recent studies have explored the possibility of attaching antimicrobial peptides (AMPs) and other antibiotics to surfaces with a view to develop surfaces that resist biofilm growth for use in medical devices and industrial structures,7-9 such as reverse osmosis (RO) membranes.10-12 AMPs are effective against a wide variety of bacteria,13 they are non-toxic to humans, and bacteria hardly acquire resistance to AMPs.14 However, less studies have been directed towards investigating the influence of the surface polymer composition on the antimicrobial activity of peptides15 and especially whether polymer compositions known to resist organic fouling or biofouling can influence antibacterial action, or synergize with the activity of the attached antibiotic. The initial adhesion of biofouling material upon a surface is a complex process where the surface charge, hydrophilicity, and chemical composition play a role. 16-17 Hydrophilic – charged polymer compositions, in particular zwitterionic polymers, are known to inhibit binding of proteins and other substances that lead to biofouling.18 The very strong hydration layer caused by the zwitterionic surface prevents adherence of proteins and other solutes.19-21 PEG-based materials have also been demonstrated to have antifouling properties.20-23 Such surface modifications with various composition and properties can be conveniently prepared using


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various types of radical polymerization reactions. For example, a “grafting from” surface reaction using redox initiators and acrylate monomers results in covalently bound polymers on surfaces with high thermal and solvent stability. This reaction is convenient and has been performed on various surfaces types in water and at room temperature.24-26 In this study the antibacterial and antifouling properties of polymer layers with various chemical compositions in combination with an antimicrobial agent were investigated, by grafting polymers with various compositions including zwitterionic, PEG, or charged together with magainin-2 onto RO membranes. Magainin-2 is an AMP with antimicrobial activity against a wide-range of bacteria, and maintains its activity when linked to surfaces.12 The peptide – grafted polymer compositions were characterized, and their relative effects on antifouling were determined by testing their abilities to inhibit the biofilm growth of P. aeruginosa. 2. Materials and Methods 2.1. Materials ESPA-HDR RO membranes were purchased from Nitto Hydranautics (Delfgauw, Netherlands). The peptide magainin-2, linked to a rink-amide resin with acid labile side chain protecting groups (peptide sequence: GMASKAGAIAGKIAKVALKAL-NH2), was purchased from peptide 2.0 (Chantilly, VA). 4-Pentyonic acid (98%) was purchased from Acros Organics (Morris Plains, NJ). Cupric sulfate, 11-Azido-3,6,9-trioxaundecan-1-amine (azide), N-(3Sulfopropyl)-N-methacroyloxyethyl-N,N-dimethylammonium betaine (SPE), 2-Hydroxyethyl methacrylate (HEMA), Polyethylene glycol methacrylate (PEGMA) MW 360 Da, Potassium metabisulfite

(K2S2O5),

Potassium

persulfate

(K2S2O8),

[2-

(Methacryloyloxy)ethyl]trimethylammonium chloride (MOETMA) solution 80% in H2O and


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hexamethyldisilazine were purchased from Sigma-Aldrich (St. Louis, MO). Gluteraldehyde 50% in water was purchased from Alfa Aesar (Boston, MA), sodium cacodylate (0.2 M, pH 7.4) and osmium tetroxide (4% aq.) solution were purchased from Electron Microscopy Sciences (Hatfield, PA). 2.2. Peptide modification Alkyne-magainin-2: The alkyne group coupling to magainin-2 was obtained by reacting 4-pentyonic acid with a dry protected magainin-2-resin (0.1 mmol) followed by deprotection and peptide cleavage from the resin as previously reported,12 except that the final alkyne-magainin-2 product was purified (> 92%) by preparative reversed-phase HPLC (Waters 600 HPLC) as described by Cohen et al.27 The preparative method involved a flow rate of 12 mL/min, a 35 min binary gradient of 0.1% TFA in water (A) and 0.085% TFA in 75% acetonitrile in water (B) (10% to 35%B). The purified alkyne-magainin-2 was analyzed by MALDI-TOF-MS and HPLC as previously described,27 and the results are shown in Figure S1, Supporting Information. 2.3. Membrane Modification Polymer grafting on membranes (Step I, Figure 1): RO membranes were modified by the redox-initiated graft polymerization reaction, as follows: Round ESPA RO membrane coupons (32 mm diameter) were first washed by the washing procedure described previously.12 Next, the membranes were immersed in 4 mL mixtures of the following monomers in DDW: PEGMA:MA, SPE:MA, MOETMA:MA, or HEMA:MA at 4:1 molar ratio in total concentration of 1 M for 7.5 min in a 6-well plate. The initiators K2S2O8 and K2S2O5 were each dissolved in DDW (0.1 M), and firstly K2S2O8 (0.5 mL) was added to the monomer solution and after 1.5 min the K2S2O5 (0.5 mL) solution was added. The RO membranes were agitated in the grafting


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solution for 20 min on a Unimax 1010 orbital platform shaker (Heidolph, Kelheim, Germany) at 25 °C. Finally, the membranes were washed 3× with water in a sonicator (10 min each wash),12 and were stored in DDW at 4 °C until further use. The four different grafted copolymers were designated as pSPE-co-pMA (a copolymer of SPE and MA), pPEGMA-co-pMA, pMOETMAco-pMA and pHEMA-co-pMA. Azide-modified membranes (Step II, Figure 1): A membrane modification by the azide group was achieved by linking 11-Azido-3,6,9-trioxaundecan-1-amine to the RO membranes or grafted RO membranes (round membranes, 32 mm diameter) by using EDC/sulfo-NHS coupling system as described previously.12 The membranes were stored in water at 4 °C. Magainin-2-modified membranes (Step III, Figure 1): The azide-modified membranes were cut into circles of 22 mm diameter. Alkyne–magainin-2 (1 mM) was dissolved in 1 mL DDW containing 1 mM cupric sulfate (CuSO4) and sodium ascorbate (2 mM), and the solution was added to the azide-modified membrane, and gently agitated for 2 h at 40 °C. The membranes were then washed with 1 mM aqueous HCl solution (2 × 10 min) and then with DDW (3 × 10 min) in a sonicator, and stored in DDW at 4 °C. 2.4. Membranes Surface Analyses Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR): The FTIR analyses of the dry membranes were done by a VERTEX 70/80 spectrophotometer (Bruker Optiks GmbH, Ettlingen, Germany).12 The analysis of each membrane was an average spectrum of six measurements that were obtained from random locations. Each measurement included 40 scans on average, at 4 cm-1 resolution, in the range of 4000-400 cm-1.


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X-ray photoelectron spectroscopy (XPS): The XPS analyses were performed as previously described on ESCALAB 250 spectrometer (Thermo Fisher Scientific, Waltham, MA).12 The signals from C1s, N1s, O1s, and S2p were detected by fixing different separated elements to the experimental data by means of nonlinear least-squares curve fitting. Surface contact angle measurement: The sessile drop-contact angle of the membranes was measured with DDW by an OCA-20 contact angle analyzer (DataPhysics Instruments, Filderstadt, Germany) by applying a water droplet (5 μL) on the dried membrane. The contact angle was calculated by the SCA-20 software (DataPhysics Instruments) where an average of six drops was calculated for each membrane.12 Zeta Potential (ζ): Zeta potential was determined by the streaming potential method using the SurPASS Electro-kinetic Analyzer (Anton-Paar KG, Graz, Austria) by tangential measuring technique in the adjustable gap cell (10 × 20 mm2 size).26 The measurements were performed at room temperature using an electrolyte of 1 mM aqueous KCl with a target ramp pressure of 380 – 400 mbar in pH range of 3 – 9 (titrations made by the addition of 0.1 M aqueous solutions of NaOH or HCl). Zeta potential values were calculated by the relationship between the measured streaming current and the zeta potential, solving the Poisson-Boltzman equation. The equation was solved via the Helmholtz-Smoluchowski equation for streaming current given in Equation 1, Equation 1. ζ =

𝑑𝐼 𝑑𝑃

×

𝜂 𝜀 ― 𝜀0

×

𝐿 𝐴

where ζ is the zeta potential, dI is the measured streaming current, dP is the applied pressure used to force the electrolyte solution to flow over the measured surfaces, ε0 is the vacuum permittivity, ε is the relative dielectric constant of the electrolyte solution (1 mM KCl), η is the dynamic


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viscosity of the electrolyte, L is the length of a rectangular slit channel formed between two planar membranes of the adjustable gap cell and A is its cross-section (A = W × H, with channel width W and gap height H). 2.5. Contact Killing Assay A single colony of P. aeruginosa was inoculated in 10 mL of LB broth and incubated overnight at 30 °C on a shaker at 150 rpm. The culture was then diluted in 5 mL LB broth (at 1:100 ratio) and was allowed to grow for about 6 h to the exponential phase (OD600 ~0.5). The cells were diluted with a sterile PBS buffer (pH 7.4) to 104 CFUs. Circular membrane coupons (16 mm diameter) were taped to a sterile glass slide with a double-sided adhesive tape and placed inside a sterile Petri dish. Then, 100 µL of bacterial culture (~4000 cells) from the diluted cells were transferred onto each of the membranes, and evenly spread on its surface by a microscope coverslip, as previously described.11, 28 The membranes were then incubated for 3 h at room temperature, the coverslips were gently removed, and the membranes, together with the coverslips, were washed with 0.5 mL of sterile PBS by continuous aspiration. The plate-count method with sterile LB agar incubated for 24 h at 30 °C was used to quantify the viable CFUs in the PBS solutions. 2.6. Biofilm Growth Assay The analysis was performed to RO membranes grafted with the four polymers, membranes modified by polymers and magainin-2, and unmodified membranes. The flow cell (31 mm × 26 mm × 36 mm) and the membranes were prepared and washed as previously described.11-12, 29 A culture of Gram-negative P. aeruginosa (PAO1 strain) were incubated and shaken overnight in LB broth growth media at 30 °C. Then, the culture was diluted to 1/100 in a


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fresh media and was incubated with shaking at 30 °C until reaching an optical density of ~ 0.8 at 600 nm (Genesys 8 spectrophotometer, Thermo Scientific, Loughborough, UK).30-31 The bacterial solution was diluted to OD600 of 0.1, and the P. aeruginosa culture (50 mL) was flowed across the membrane at 2.5 mL/min using a peristaltic pump as described previously.12 The experiments were run for 36 h at 2 mL/min at room temperature. The membrane-attached coverslips were then removed, and the bacteria were stained. The staining procedure was performed as described previously.12 The biofilm visualization was done using confocal laser scanning microscopy (CLSM; Zeiss LSM 510 META) with a dry objective Plan-NeoFluar (20 × magnification, 0.5 numerical aperture). Data were analyzed using the Imaris 3D imaging software (Bitplane, Zurich, Switzerland), and quantitative analysis (biofilm volume and average thickness) was conducted using COMSTAT on Matlab.32 Average values of five representative images are reported. 2.7. Scanning Electron Microscopy (SEM) RO membranes (modified and unmodified) attached to coverslips were exposed to diluted culture solution of P. aeruginosa followed by flow of a growth media at 30 °C for 36 h under the same conditions that are described in section 2.6. The membrane-attached coverslips were then removed, and an excess electrolyte solution was carefully removed with a fine paper. The membranes were immersed in 0.05 M sodium cacodylate buffer (pH 7.4) complemented with 2% gluteraldehyde (2 mL 8% gluteradehyde, 2 mL 0.2 M sodium cacodylate buffer, and 4 mL DDW) for 1 h. Then the membranes were immersed for 10 min in 0.05 M sodium cacodylate buffer, and rinsed twice with the same buffer. In the second step the membrane samples were immersed in 0.05 M sodium cacodylate buffer complemented with 1% osmium tetroxide (2 mL osmium tetroxide (4%), 2 mL 0.2 M sodium cacodylate buffer and 4 mL DDW) for 1 h. The


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membranes were immersed for 10 min in 0.05 M sodium cacodylate buffer, then rinsed twice with the buffer. Membranes were dehydrated in ethanol/water solutions with increasing ethanol concentrations (25, 50, 75, 95, and 100%) during a 20-min incubation period. Samples were washed once with hexamethyldisilazine at 4 °C and dried overnight in a hood at room temperature. The dry membranes were coated with gold and analyzed by SEM (JSM-7400F, JEOL, Tokyo, Japan). 3. Results and Discussion 3.1 Antimicrobial Surface Preparation and Characterization Magainin-2 functionalized with an alkyne group at its N-terminus was synthesized similar to previously published procedures, purified by RP-HPLC and the identity confirmed with MALDI-TOF mass spectrometry (see Materials and Methods, and Figure S1, Supporting Information). This alkyne peptide was then reacted with a series of RO membranes grafted with polymers of various compositions that were functionalized with azide groups (Figure 1). Specifically, the series of membranes included hydrophilic polymer coatings on RO membranes, prepared via grafting from the surface with the following monomers: SPE (zwitterionic), HEMA (neutral), PEGMA (neutral-PEG) and MOETMA (positively charged). MA (methacrylic acid) contains a carboxylic acid functionality and was included in each of the monomer solutions in order to attach the amine-azide bifunctional linker to the grafted polymer coating via activation of the carboxylic acid with EDC/sulfo-NHS.


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O H3C

R O

CH2 HO O H3C

O

OH

mO

CH2

O

MA

(1)

RO membrane

C O m

O

R

O

H2N

3

N3

O 3

N3

(2)

EDC, Sulfo-NHS

K2S2O8, K2S2O5 k

H N C O m O R C O k

k

H N O

C O

O 3

N N N

O

R

(3)

CuSO4, Ascorbic acid H3C CH3 N+

O R: O O

O

O

n

OH

O OS O

(SPE) (PEGMA)

H3C CH3 N+ CH3

(MOETMA)

OH

(HEMA)

magainin-2

Figure 1. Magainin-2 attachment onto RO membrane surfaces. (1) graft polymerization using MA:monomer at 1:4 molar ratio with SPE, PEGMA, MOETMA, HEMA as monomers; (2) azide attachment reaction using 60 mM 11-Azido-3,6,9-trioxaundecan-1-amine, 20 mM sulfo-NHS, and 20 mM EDC in a sodium phosphate buffer (0.1 M, pH 7.4) at room temperature, overnight; (3) click chemistry (CuAAC) reaction using alkyne-magainin-2 (1 mM), CuSO4 (1 mM), and sodium ascorbate (2 mM) at 40 °C for 2 h. FTIR measurements were performed to the membrane surfaces after each reaction step. For example, the pSPE-co-pMA modified surface (Figure 1 step 1) showed new absorbances at 1716 cm-1 and 939 cm-1, assigned to the ester-carbonyl and to the quaternary ammonium group, respectively. Also, the peak at ca. 1037 cm-1 indicated the sulfonate functionality on the surface


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(Figure 2, A).33-34 Similarly, for the surfaces consisting of pHEMA-co-pMA, pPEGMA-co-pMA, and pMOETMA-co-pMA the IR spectra (Figure S2 A-F, Supporting Information) also showed new additional peaks at 1716 cm-1, 1037 cm-1 and 939 cm-1, that were assigned to the estercarbonyl of pHEMA-co-pMA, to the C–O ether vibration of PEG in pPEGMA-co-pMA ,23, 35 and to the quaternary ammonium group of pMOETMA-co-pMA (Figure S2 A-F, Supporting Information). Next, alkyne-magainin-2 was covalently attached to the azide-functionalized surface via a click chemistry reaction. The absorption signal of the amide bond on the surface shifted from 1646 cm-1 to 1656 cm-1 with an increased intensity. The amide peak of the peptide bond at 1656 cm-1 was normalized to the polysulfone peak at 1487 cm−1 and the resulting peak ratio increased from 0.9 for the membranes without a peptide to 1.3 for all membranes linked to magainin-2 peptide (Figure 2, B and Figures S2 B,D,F,H, Supporting Information).36 In addition, after the attachment of the peptide, there was a significant decrease in the peaks at 1716 cm-1, 1037 cm-1, and 939 cm-1 compared to all grafted membranes from the previous steps (Figure 2, A and Figure S2 A,C,E,G, Supporting Information), due to the presence of an additional (peptide containing) layer on the surface, which mask those absorption peaks. The XPS analysis on membrane samples before and after the peptide attachment step showed that the C/N ratio decreased from ~11 to ~7 due to the presence of peptides which possess higher nitrogen content than the methacrylate polymers (Table S1, Supporting Information). Since SPE zwitterion contains a sulfonic acid group, increased intensity at the sulfur binding energy of ~168 eV was observed compared to the unfunctionalized membrane (Figure 3, A). The smaller sulfur peak of the unmodified membrane (at ~168 eV) results from the sulfone group in the polysulfone layer of the membrane. The peptide modified membranes gave rise to a new peak at 164 eV due to the thio-ether sulfur in methionine residue of magainin-2


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(Figure 3 B) and the same was observed in the other peptide functionalized membranes (Figure S3 A, Supporting Information). Also, a new peak at ~402.7 eV was observed in both the pSPEco-pMA and the pMOETMA-co-pMA surface modifications due to the presence of a quaternary ammonium group. However, after peptide linked to the SPE/MA, the quaternary ammonium peak was not observed. Since XPS analysis measures to only a surface depth of 10 nm, a thick peptide coating might have decreased the signal (Figure S2 B, Supporting Information).


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Figure 2. A) FTIR spectra of RO membranes modified via the SPE grafting, at all three stages: Grafting of pSPE-co-pMA, pSPE-co-pMA with azide, and pSPE-co-pMA linked to magainin-2. B) Amide peak ratios (1656 cm-1/1487 cm-1) of magainin-2 linking calculated from the FTIR spectra.


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Figure 3. XPS analysis showing S2p of unmodified membrane and a membrane grafted with pSPE-co-pMA (A) and the S2p of magainin-2 linked to pSPE-co-pMA surface (B). The zeta potential of the membrane-grafted polymers were analyzed and compared with the zeta potential values of the peptide modified membranes in the pH range of 3 to 9 (Figure 4). All grafting mixtures included MA, therefore mixtures composed of zwitterionic or uncharged monomers might lead to surfaces that are more negatively charged compared with the unmodified membrane. On the other hand, magainin-2 is a highly positively charged peptide, and is expected to lead to surfaces that are more positively charged. Also, the polyamide membrane surfaces are negatively charged probably due to many unreacted carboxylic acid groups that are


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formed in the interfacial polymerization reaction. Indeed, we observed that membranes composed of MA with neutral monomers gave a slightly more negatively charged surface. Grafting with pMOETMA-co-pMA led to a more positive charge between pH 3 to 5.5 and from pH 5.5 to 9.5 was more negatively charged compared to the unmodified surface (Figure S4, Supporting Information). The azide coupling step should reduce the number of free carboxylic acid groups and we observed a less negative surface for the azide modified pHEMA-co-pMA surface (Figure 4 B). The charge on the surfaces might play a role in facilitating bacterial attachment.37-39 For example, negative charge can repel bacteria due to the net negative charge maintained on the outer leaflet of the bacterial cell membrane. However, highly charged surfaces are sometimes more susceptible to organic fouling, which pre-conditions the surface and can enhance bacterial attachment. For peptide coated surfaces, the zeta potential was higher and was closest to 0 at pH ca. 7, which reflects the linking of the cationic magainin-2 peptide. Next, the sessile drop contact angle showed that all membranes were hydrophilic but slight differences were seen especially between peptide coated and uncoated samples (Figure 5). The unmodified membrane had a low contact angle of 26° ± 6.8 indicating a hydrophilic surface. The grafting steps and the azide functionalization steps resulted in minor changes to the hydrophilicity, ranging for example from the most hydrophilic pPEGMA-co-pMA (21° ± 2.9) to the pMOETMA-co-pMA surface coating with a contact angle of 35°. In all cases, the magainin-2 linking increased the contact angle significantly which might indicate that upon drying the membrane peptide exposes its hydrophobic face to the air, and its hydrophilic face to the membrane. For example, magainin-2 linked to the zwitterionic polymer SPE/MA gave the lowest change in contact angle (39° ± 3.5), possibly indicating that the peptide orientation or


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aggregation state is different compared to the peptide on the pPEGMA-co-pMA surface, which exhibited a contact angle of 63° ± 5.1.

Figure 4. Zeta-potential curves in the pH range of 3 – 9 of unmodified membrane, grafted membranes and membranes with polymers linked to magainin-2. A) pSPE-co-pMA modifications; B) pHEMA-co-pMA modifications, and pHEMA grafted without pMA (HEMA at 0.8 mM), and pMA grafted without pHEMA (MA at 0.2 mM).


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Figure 5. Sesile water drop contact angle values of the unmodified membranes and the grafted membranes with pSPE-co-pMA, pPEGMA-co-pMA, pMOETMA-co-pMA and pHEMA/-copMA after each step in the magainin-2 linking (Figure 1). 3.2 Bacterial Inhibition on RO Surfaces AMPs covalently bound to surfaces have demonstrated the ability to remain active against bacteria on RO surfaces,10-11 and activity parameters have been discussed.12 Here we tested whether the polymer surface composition affected the killing activity of the peptide magainin-2. For example, zwitterionic surfaces known to resist binding of foulants or bacteria might affect the peptide activity where some interaction of the bacteria with the surface is necessary.19, 22, 33, 40-43 Thus, we tested the surfaces killing properties against P. aeruginosa. After incubation of the bacteria on the surfaces, surfaces were washed and the number of CFUs obtained from the test membranes were normalized to the number of CFUs seen from the unmodified membrane. In all cases CFU counts were lower in samples from the peptide


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modified membranes in comparison to unmodified membranes (Figure 6). Those experiments tested the killing activity of the surfaces which was only affected by the peptides, where the polymeric linkers had no influence or killing activity. Due to that the only significant killing ability was available when the peptide was linked to the surface. The polymeric linkers did not interfere with the magainin-2 killing activity; hence in all the linking setups the peptide was active.

Figure 6. Bacterial growth of P. aeruginosa on RO membranes normalized to the unmodified membrane: Membranes modified with the four copolymer linkers, compared with the magainin-2 directly modified membranes. 3.3 Biofilm Growth Inhibition The biofilm growth of P. aeruginosa was also affected by the modification type using a flow cell assay. The surfaces were exposed to a flow of the bacteria culture for 20 min and then exposed


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for 36 h to a flowing nutrient broth; for testing the initial bacteria growth step.11-12,

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Subsequently, the biomass and the thickness of the biofilm was estimated by quantification of both dead and live bacteria using CLSM. These analyses indicated that the biofilms formed on the surfaces of all membranes modified with magainin-2 were thinner than those formed on the surfaces of the unmodified membranes and the polymer-only surfaces (Figure 7). For the magainin-2 grafted surfaces, the biofilm volume was reduced by up to ~ 95% as compared to the unmodified membranes. A reduction of biofilm volume by 73% was seen on membranes modified only with magainin-2, indicating that the polymer coatings in combination with the peptide led to an enhanced anti-biofouling effect. The enhanced effect may be explained by the combination of the killing activity of magainin-2 and the anti-adherence effects of the hydrophilic polymer coatings. Without peptide modification, the zwitterionic surface (pSPE-copMA), the PEG surface (pPEGMA-co-pMA; Figure 7), and the positively (pMOETMA-copMA) surface (Figure S5, Supporting Information) also gave a reduction in biofouling but less pronounced than the reduction of polymers in combination with magainin-2. In the pHEMA-copMA there were almost no reduction comparing to the unmodified membranes.


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Figure 7. The biovolume expressed as dead and live bacterial cells of a P. aeruginosa biofilm on RO membranes under flow conditions for 36 h. Unmodified RO membranes are compared with grafted membranes (pSPE-co-pMA, pPEGMA/-co-pMA, pHEMA/-co-pMA), and membranes modified

by

magainin-2,

pPEGMA-co-pMA-magainin-2,

pSPE-co-pMA-magainin-2,

pMOETMA-co-pMA-magainin-2 and pHEMA-co-pMA-magainin-2. SEM analysis of the membranes after 36 h under flow cell conditions was consistent with the trend shown by the CLSM analysis. The unmodified membrane, the zwitterionic (pSPE-copMA), the positively charged (pMOETMA-co-pMA) and the hydroxyl-bearing (pHEMA-copMA) membranes had a large amount of biofilm, (Figure 8, left) compared with the membranes modified with AMP magainin-2 (Figure 8, right). Membranes grafted with only PEG (pPEGMAco-pMA; no peptide) also showed reduced biofilm. A comparison between the grafted surfaces with the peptide on grafted surfaces revealed the decrease in amount of biofilm on the surfaces modified with magainin-2 (Figure 8). In general, the linking of the peptide dramatically reduced


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the biofilm that formed on the membrane surfaces during flow conditions. This can be seen clearly at a lower magnification (×2000) where large parts of the membrane can be viewed, showing less biofilm on the surfaces modified with the AMP (Figure S6, Supporting Information).

Figure 8. SEM images of P. aeruginosa biofilm on RO membranes after flow conditions for 36 h, at low and high magnifications (×20,000 (Low) and ×50,000 (High)). A) Unmodified


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membranes; B) membrane-magainin-2; C) pSPE-co-pMA; D) pSPE-co-pMA-magainin-2; E) pPEGMA-co-pMA;

F)

pPEGMA-co-pMA-magainin-2;

G)

pMOETMA-co-pMA;

H)

pMOETMA-co-pMA-magainin-2; I) pHEMA-co-pMA; J) pHEMA-co-pMA-magainin-2. Scale bar in low resolution panels – 2 m and in high resolution panels – 1 m. Conclusions The effect of surface grafted polymer compositions combined with the antimicrobial agent magainin-2 on RO membrane surfaces was tested. The polymer coatings were achieved by graft polymerization of methacrylic monomers, including a methacrylic acid which increased the surface concentration of carboxylic acid groups on the RO membrane. The ATR-FTIR, water drop contact angle, XPS and ζ potential analyses all pointed to the effects of the polymer coatings on the surface and the successful attachment of the peptide to the surfaces. The polymer coatings on the surface in combination with the peptide magainin-2 increased the surface antifouling properties. In all cases, less biofilm was seen on surfaces that were functionalized with both polymers and the peptide. Differences in biofilm inhibition with polymer coatings alone were seen in flow conditions assays, and the various polymer compositions in all cases increased the potency of the anti-biofouling effect. This study reveals that different antifouling strategies can be combined in order to enhance the effect, and that antimicrobial peptides might be broadly applicable on many surface types for antimicrobial surfaces or to delay biofilm growth. Acknowledgments We thank the Israel Science Foundation (grant No. 1474-13 to CJA) for financial support. NSK is grateful to the Pratt Foundation for a Ph.D. scholarship. CJA wishes to thank the


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Canadian Associates of Ben Gurion University of the Negev (CABGU) Quebec Region for support.

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Table of Contents Graphic


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Grafted Polymer Coatings Enhance Fouling Inhibition by an

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