Arginine-Based Polymer Brush Coatings with Hydrolysis-Triggered

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Arginine-based Polymer Brush Coatings with Hydrolysis-Triggered Switchable Functionalities from Antimicrobial (Cationic) to Antifouling (Zwitterionic) Gang Xu, Xianneng Liu, Peng Liu, Dicky Pranantyo, Koon Gee Neoh, and En-Tang Kang Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01000 • Publication Date (Web): 15 Jun 2017 Downloaded from http://pubs.acs.org on June 20, 2017

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Arginine-based Polymer Brush Coatings with Hydrolysis-Triggered Switchable Functionalities from Antimicrobial (Cationic) to Antifouling (Zwitterionic)

Gang Xu, Xianneng Liu, Peng Liu, Dicky Pranantyo, Koon-Gee Neoh, En-Tang Kang* Department of Chemical and Biomolecular Engineering National University of Singapore 4 Engineering Drive 4, Kent Ridge, Singapore 117576

* To whom correspondence should be addressed E-mail: [email protected] (E.T.K)

1 ACS Paragon Plus Environment

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Abstract Arginine polymer-based coatings with switchable properties were developed on the glass slides (GS) to demonstrate the smart transition from antimicrobial (cationic) to foulingresistant (zwitterionic) surfaces. L-arginine methyl ester-methacryloylamide (Arg-Est) and Larginine-methacryloylamide (Arg-Me) polymer brushes were grafted from the GS surface via surface-initiated reversible addition-fragmentation chain-transfer (SI-RAFT) polymerization. In comparison to the pristine GS and Arg-Me graft polymerized GS (GS-Arg-Me) surfaces, the Arg-Est polymer brushes-functionalized GS surfaces exhibit a superior antimicrobial activity. Upon hydrolysis treatment, the strong bactericidal efficacy switches to good resistance to adsorption of bovine serum albumin (BSA), the adhesion of gram-positive bacteria S. aureus and gram-negative bacteria E. coli, as well as the attachment of Amphora coffeaeformis. In addition, the switchable coatings are proven to be biocompatible. The stability and durability of the switchable coatings are also ascertained after exposure to filtered seawater for 30 days. Therefore, deposition of the proposed “smart coatings” offers another environmentally-friendly alternative for combating biofouling.

Keywords: Surface modification, arginine, switchable coatings, antifouling, antimicrobial, and SI-RAFT polymerization


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1. Introduction Biofouling has been of great concern as an economic and ecologic problem in recent years.1-5 The unwanted accumulation and settlement of micro- and macro- organisms on surfaces can have serious consequences, including but not limited to operational failure in medical surgery,6,7 increased fuel consumption of marine vessels,8 accelerated ageing process of underwater sensors9 and reduced service lifetime of marine structures.10-12 According to the latest statistic report of International Maritime Organization (IMO), the annual cost spent on marine shipping without antifouling protection is about $150 billion more than that on efficient-antifouling marine shipping.13

To prevent the initial adhesion and attachment of the micro/macro- foulers, diverse strategies have been developed.11,14-17 Notably, surface modification by tethering of functional coatings is widely accepted as an effective method.9,15,17 Taking only the environmentally friendly coatings into consideration, these coatings can generally be classified into two categories: polymer coatings with fixed functionalities18,19 and polymer coatings with switchable functionalities.20-22 The latter type of polymer coating allows the transition from one specific functionality to another with additional stimulus, and offers possibilities and opportunities to design an intelligent “smart coating” to confer substrates with multiple functionalities. For example, a novel monomer which can transform reversibly between an open carboxylate state and a six-membered lactone ring state, allows switching between antibacterial and antifouling capabilities driven by either acidic or basic conditions.20

To date, surface-initiated reversible addition-fragmentation chain-transfer (SI-RAFT) polymerization has been applied as an effective approach to deposit polymer brushes on various substrates.23-25 Moreover, the SI-RAFT polymerization technique is attractive, not


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only because of the mild reaction conditions without the requirement of metal catalysts,26,27 but also of the increased brush density via the “grafting from” over the “grafting to” method.28,29

On the other hand, zwitterionic polymers have attracted extensive attention due to their wide spread applications ranging from biomedical drug delivery to engineering materials.30-34 Specifically, zwitterionic polymer brushes are commonly employed to combat surface adhesion and fouling, since the electrostatically induced hydration exhibits superior resistance to non-specific protein adsorption, bacterial adhesion, and subsequent biofilm formation.11,20,35 As a novel member of zwitterionic molecules, arginine and its derivatives are mainly used as nontoxic carriers in drug delivery systems,32 or cell-penetration enhancers in therapeutic systems.36 In addition to existing applications, it will be important and desirable to extend the potential applications of arginine and its derivatives. Accordingly, a systematic study on the anti-adhesion efficacies of arginine-based zwitterionic polymer brushes is carried out. Inspired by the concept of “smart coating”, it will also be interesting to develop an arginine-based switchable coating and to evaluate its potential performance against diverse micro- and macro-foulers.

In addition to the reported applications of arginine in drug delivery and therapeutic systems, we have broadened the application range of arginine and its derivatives to antifouling and antimicrobial surface coatings. In this study, we fabricated an arginine-based polymer brush coating by SI-RAFT polymerization, in which functionality transition from antimicrobial to antifouling was achieved by a simple hydrolysis treatment, as illustrated in Scheme 1. Mussel-inspired polydopamine (PDA) was utilized as the anchor layer on glass slide (GS) surface via catechol coordination,37 followed by functionalization of chain transfer agents


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(CTS) on the GS-PDA surface. Subsequently, SI-RAFT polymerization was carried out using the newly-synthesized Arg-Est as monomer, to yield the antimicrobial GS-Arg-Est surface. The GS-Arg-Est surface can be hydrolyzed to achieve property transition from antimicrobial (cationic) to the antifouling (zwitterionic), since hydrolysis of the ester linkages regulates the charges of assembled polymer brushes.38,39 For comparison purpose, the zwitterionic GSArg-Me surface was also prepared via SI-RAFT polymerization of the Arg-Me as monomer. The antimicrobial and antifouling efficiencies of the resulting polymer brushes-coated GS surfaces were examined by the adsorption of bovine serum albumin (BSA), the adhesion of both gram-negative bacteria E. coli. and gram-positive bacteria S. aureus, and the attachment of Amphora coffeaeformis. In addition, the cytotoxic effect of the resulting coating was also investigated with 3T3 fibroblasts in MTT assays.

2. Experimental Section 2.1 Materials Dopamine hydrochloride (98%), tris(hydroxymethyl) aminomethane hydrochloride (TrisHCl),

4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic

acid

(HEPES),

4’-azobis(4-

cyanovaleric acid) (ACVA), 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, 98%), 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid N-succinimidyl ester, Larginine monohydrochloride, L-arginine methyl ester dihydrochloride, methacrylic anhydride and albumin-fluorescein isothiocyanate conjugate (BSA-FITC, product no. A9771) were purchased from Sigma-Alrich Chemical Co., St. Louis, MO. All other solvents and reagents were purchased from either Sigma-Aldrich or Merck Chem. Co., and were used as received. Gram-negative bacteria strain of Escherichia coli (E. coli, ATCC, 14948) and gram-positive bacteria strain of Staphylococcus aureus (S. aureus, ATCC 12228) were obtained from


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American Type Culture Collection, Manassas, VA. The LIVE/DEAD BacLight Bacterial Viability Kit L131152 was purchased from Molecular Probes Inc., Eugene, OR.

2.2 Synthesis of L-arginine-methacryloylamide (Arg-Me) and L-arginine methyl estermethacryloylamide (Arg-Est) L-arginine-methacryloylamide (Arg-Me) was synthesized according to the procedures published previously.36 Briefly, 2.1 g of L-arginine monohydrochloride (10 mmol) was dissolved in 20 mL of saturated NaHCO3 solution and the resulting mixture was cooled to 0 °C. Next, 1.5 mL of methacrylic anhydride (10 mmol) was added drop-wise under vigorous stirring, and the reaction was allowed to proceed for 2 h. Subsequently, the pH value of the mixture was adjusted to 1 using hydrochloric acid, saturated with sodium chloride, filtered, washed with 30 mL of ethyl acetate three times, and then extracted using 20 mL of 1:1 vol/vol mixture of ethyl acetate/isopropanol for three times. The ethyl acetate/isopropanol fractions were concentrated under reduced pressure, and filtered. The resulting colorless oil was dissolved in 50 mL of deionized water and lyophilized. L-arginine methyl estermethacryloylamide (Arg-Est) was synthesized similarly, except without the acidification step. 1

H NMR (600 MHz, Arg-Me in D2O): δ (ppm) = 5.75 (s, 1H, HHC=C), 5.53 (s, 1H, HHC=C),

4.46 (dd, J = 9.2, 5.1 Hz, 1H, α-CH), 3.24 (t, J = 6.8, 2H, δ-CH2), 1.96 (s, 3H, -CH3), 1.87– 1.71 (m, 2H, γ-CH2), 1.71–1.52 (m, 2H, β-CH2). 1

H NMR (600 MHz, Arg-Est in D2O): δ (ppm) = 5.75 (s, 1H, HHC=C), 5.53 (s, 1H, HHC=C),

4.49 (dd, J = 9.2, 5.1 Hz, 1H, α-CH), 3.78 (s, 3H, -OCH3), 3.24 (t, J = 6.8 Hz, 2H, δ-CH2), 1.95 (s, 3H, -CH3), 1.88–1.70 (m, 2H, γ-CH2), 1.70–1.51 (m, 2H, β-CH2).

2.3 Preparation of the polymer brush coatings 2.3.1 Immobilization of dopamine anchor on glass slide


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Pristine GS coupons of 1× 1 cm2 in area were prepared, washed and activated according to the methods published previously.40 These pristine GS coupons were immersed in 30 mL of Tris-HCl solution (10 mmol, pH 8.5) containing 60 mg of dopamine hydrochloride in a petri dish. The petri dish was placed in a water bath shaker at 37 °C for 5 h. The resulting GSpolydopamine (GS-PDA) substrates were washed with deionized water, and dried under reduced pressure.

2.3.2 Preparation of GS-PDA/CTS Chain transfer agents were functionalized on the GS-PDA surface for the subsequent SIRAFT polymerization according to the previous method with some modification.41 Briefly, 4Cyano-4-(phenylcarbonothioylthio) pentanoic acid N-succinimidyl ester (CTS, 90 mg, 0.3 mmol) was dissolved in 20 mL of methanol with trimethylamine (56 µL, 0.4 mmol) at room temperature. Subsequently, 10 coupons of GS-PDA were introduced into the solution and the mixture was degassed for 1 h under a nitrogen atmosphere, sealed and agitated in an orbital shaker for 48 h. The obtained CTS-modified GS coupons, denoted as GS-PDA/CTS, were washed with methanol and deionized water, and then dried under reduced pressure.

2.3.3 Surface-initiated RAFT polymerization The grafting of polymer brushes was carried out via surface-initiated reversible addition fragmentation chain transfer (SI-RAFT) polymerization. Four coupons of GS-PDA/CTS, Arg-Est (2.56 g, 10 mmol) and ACVA (0.0020 g, 0.012 mmol) were introduced into a mixture of 10 mL of deionized water in a conical flask. The mixture was stirred and degassed with purified argon for 45 min. The flask was sealed and placed in an oil bath maintained at 70 °C for 24 h. After the polymerization, the Arg-Est polymer-grafted substrates, denoted as GS-Arg-Est, were collected, washed thoroughly with deionized water, and dried under


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reduced pressure. The Arg-Me polymer-grafted substrates, denoted as GS-Arg-Me, were also prepared as a control via SI-RAFT polymerization. In addition, the hydrolyzed GS-Arg-Est surface was prepared by immersing the GS-Arg-Est surface in PBS solution (pH 10.0) at 37 °C for 15 h.

2.4 Antimicrobial and antifouling assays 2.4.1 SPR measurements of protein adsorption A custom-built surface plasma resonance (SPR, Biacore 2000 system, Biacore AB, Sweden) sensor was used to measure the real-time protein adsorption. The processes for preparing polymer brush coatings on Au SPR chip surfaces was the same as that on the GS substrates described above. For the adsorption assays, the pristine and polymer-functionalized Au SPR chips were exposed to a flow (0.05 mL/min) of 0.1 mg/mL BSA protein in HEPES buffer (pH 7.4) for 10 min, after which the sensor chips were rinsed with HEPES for 5 min. The SPR-based protein adsorption experiments on each chip were repeated three times to validate their anti-adhesion performances.

2.4.2 Bacterial adhesion assays Gram-negative bacteria E. coli and gram-positive bacteria S. aureus were used to test the antimicrobial and antifouling performance of the pristine and polymer-functionalized GS surfaces. Bacteria were cultured, centrifuged, washed and finally suspended at a concentration of 107 cells/mL according to the method reported previously.35 One mL of the bacterial suspension was added to cover the 1 × 1 cm2 sample surface in a 24-well plate (Nalge, Nunc Int., Rochester, NY) under static condition at 37 °C for 4 h. After that, the loosely adhered bacteria on the samples were removed by washing thrice with ultrapure water. Each sample substrate was then stained with LIVE/DEAD BacLight solution for 15 min. The


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samples were subsequently observed under the Nikon fluorescent microscope (Nikon ECLIPSE Ti-U fluorescent microscope, Tokyo, Japan). Green filter (excitation/emission: 450 nm-490 nm/500 nm-550 nm) and red filter (excitation/emission: 510 nm-560 nm/605 nm-685 nm) were used to observe the live and dead bacteria, respectively. For each GS surface, three sample locations were selected, and the fluorescence images of each sample were captured randomly using the Nikon Digital Sight DS-U3. For quantitative analysis, ImageJ software (http://imagej.nih.gov.libproxy1.nus.edu.sg/ij/) was used to analyse the images obtained from microscopy. The mean value of bacterial adhesion fraction was determined from the three fluorescence images of each GS surface. Anova analysis for sets of data was used for pairwise comparison between the pristine and polymer-functionalized surfaces.

2.4.3 Amphora attachment assays Amphora coffeaeformis (UTEX B2080) was cultured, collected and diluted to a concentration of 105 cells/mL prior to use as described in the literature.42 For the settlement test, each sample substrate of 1 × 1 cm2 in size was immersed in 1 mL of Amphora suspension at room temperature for 24 h. The coupons were then washed three times with deionized water to remove the loosely adhered or unattached cells. The Amphora cells attached on the coupons were studied with a Nikon Eclipse Ti microscope, equipped with an excitation filter of 535 nm and an emission filter of 617 nm. Quantification of the adhered Amphora cells was carried out by ImageJ software. All experiments were performed in triplicate with three samples to obtain the mean values. The results were expressed as percentages relative to the cell number obtained from the pristine GS surface (100%). Anova analysis for sets of data was used for pairwise comparison between the pristine and polymer-functionalized surfaces.

2.5 Cytotoxicity assays


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Cytotoxicity of the pristine, GS-Arg-Me, GS-Arg-Est and hydrolyzed GS-Arg-Est substrates were assayed with 3T3 fibroblasts (ATCC, Manassas, USA) via MTT assays, following the procedures reported previously.43 The pure Dulbecco’s Modified Eagle’s Medium (DMEM) medium without pre-treatment was chosen for the control experiments.

3. Results and Discussion 3.1 1H NMR characterization of Arg-Me and Arg-Est monomers 1

H NMR spectroscopy measurements were performed to verify the successful synthesis of

Arg-Me (top) and Arg-Est (bottom), as shown in Figure 1. The chemical shift at 3.24 ppm (J = 6.8 Hz) is attributed to the δ-CH2 of arginyl moieties.44 The characteristic signals at 5.75 and 5.53 ppm represent the methacrylic functionality, consistent with those reported methacrylate-modified monomers.45 Due to the similar chemical structure between Arg-Me and Arg-Est, their 1H NMR spectra almost overlapped, except for the newly-appeared signal with a chemical shift at 3.9 ppm. The new signal is in good agreement with the additional methyl group in the Arg-Est molecule.

3.2 XPS characterization of the polymer-coated GS surfaces Figure 2 shows the XPS wide-scan, C 1s and N 1s core-level spectra of the polymerfunctionalized GS surfaces. In the XPS wide-scan spectrum of the GS-PDA/CTS surface (Figure 2a), two apparent XPS signals with respective binding energy (BE) at 165 and 228 eV are attributed to the S 2p and S 2s signals of thiol groups,46,47 suggesting the covalent bonding of chain transfer agents. In Figure 2b, the XPS C 1s core-level spectra of the GSPDA/CTS surface can be curve-fitted into five peak components with BEs at 284.6, 285.6,


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286.2, 287.8 and 288.7 eV, attributable to the C-H, C-N, C-O, C=O and O-C=O species, respectively.46,48 The XPS N 1s core-level spectrum (in set), with respective signal at the BE of 399.7 and 401.5 eV are associated with the amine ((C)-N-) and protonated amine (-NH3+) species.40,45 After SI-RAFT polymerization of Arg-Me on the GS-PDA/CTS surface, the intensity of O-C=O species in the C 1s core-level spectrum of the GS-Arg-Me surface (Figure 2d) increases, consistent with the formation of Arg-Me polymer brushes via the ‘grafting from’ method. Similarly, the intensity of O-C=O species in the C 1s core-level spectrum of the GS-Arg-Est surface also increases (Figure 2f). After a 15 h of hydrolysis treatment in PBS (pH 10.0), the line-shape of deconvoluted C 1s core-level spectrum of the hydrolyzed GS-Arg-Est surface (Figure 2h) remains similar to that of the GS-Arg-Me and that of the GS-Arg-Est surfaces, indicating that 15 h of hydrolysis under alkaline condition has negligible effect on the chemical structure of the GS-Arg-Est surface.

3.3 Arginine-based polymer brush coating’s switchable functionalities 3.3.1 Static water contact angles The static water contact angles of the pristine and polymer-functionalized GS surfaces were determined to investigate the change in surface wettability against modification and hydrolysis. The pristine GS surface presents a small contact angle of 38°, while the surface becomes hydrophobic with a water contact angle of 50° after the modification with the PDA/CTS layer. SI-RAFT polymerization of zwitterionic Arg-Me polymer brushes significantly reduces the water contact angle from 50° to 17°, indicating a strong electrostatic interaction with water molecules.30 In contrast, deposition of Arg-Est polymer brushes increases the surface hydrophilicity only slightly with a water contact angle of 34°. Hydrolysis treatment on the GS-Arg-Est surface increases the surface wettability further to give a smaller water contact angle of only 23°, which can be explained by the partial


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formation of zwitterionic Arg-Me polymer brushes upon hydrolysis of the Arg-Est polymer brushes.

3.3.2 Surface charges In Figure 4, the zeta potential of the pristine and polymer-functionalized GS surfaces was measured to monitor the change in surface property after modification and hydrolysis. For all surfaces, the zeta potential decreases accordingly with increase in pH from 5.0 to 10.0. For instance, the pristine GS surface is negatively charged over this pH range. The GS-PDA/CTS surface exhibits negative surface charge when pH is higher than 6, with a zeta potential of about -28 mV at pH 7.4. The increase is attributable to the coated polydopamine anchor layer, as well as the chain transfer agents. SI-RAFT polymerization of zwitterionic Arg-Me further increases the surface charge, the GS-Arg-Me surface is positively charged at low pH and negatively charged at high pH, with an isoelectric point close to pH 7. In contrast, the GSArg-Est surface is positively charged from pH 5 to 10, which is in good agreement with the positive guanidine groups in the grafted Arg-Est polymer brushes. However, after alkaline hydrolysis treatment, the hydrolyzed GS-Arg-Est surface represents a neutral zeta potential, which is close to the surface charge distribution of the GS-Arg-Me surface. The shift of surface charge from ~43 mV (the GS-Arg-Est surface) to -1 mV (the hydrolyzed GS-Arg-Est surface) upon hydrolysis confirms the hydrolysis of ester groups in the Arg-Est polymer brushes, which is consistent with the result obtained from water contact angle measurement.

3.3.3 Surface morphologies The root-mean-square roughness (Rq) over an area of 3 × 3 µm2 was measured to investigate the surface morphologies of functionalized surfaces (Figure 5). Compared to the GSPDA/CTS surface, both the GS-Arg-Me and GS-Arg-Est surfaces show smoother surface


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after SI-RAFT polymerization since the Rq value decreases from 1.81 nm to 0.67 nm for the GS-Arg-Me surface and 0.84 nm for the GS-Arg-Est surface. In addition, the hydrolyzed GSArg-Est surface exhibits a smoother surface with a Rq value of 0.76 nm. It can be assumed that arginine-based zwitterionic polymer brushes have a smaller Rq value induced by the noncovalent electrostatic attraction between negatively charged −COO− groups and positively charged −NH3+ groups.49

3.4 Antifouling and antimicrobial evaluations 3.4.1 Protein adsorption tests Protein adsorption has a significant effect on the adhesion of fouling organism since it can adversely affect the surface functionalities.50 In Figure 6, BSA adsorption on the pristine and polymer-functionalized GS surfaces was studied using the surface plasma resonance (SPR) technique. Obviously, the pristine Au chip exhibits the highest adsorption of BSA, with the highest response of approximately 1824 RU after 10 min of BSA injection and 5 min of washing with HEPES. The Au-PDA/CTS surface also presents a high protein adhesion at ~1600 RU. After the SI-RAFT polymerization of Arg-Me, the Au-Arg-Me surface displays the best anti-adhesion performance for BSA protein adsorption as it has the lowest response of about 100 RU. The Au-Arg-Est surface also exhibits promising resistance to BSA protein with a response of ~560 RU. The hydrolyzed Au-Arg-Est surface exhibits a better antifouling efficiency (about 210 RU) in comparison to that of the Au-Arg-Est surface, indicating that the anti-adhesion efficacy is further enhanced on the zwitterionic surface.

3.4.2 Bacteria adhesion assays Bacterial adhesion on the artificial surfaces is considered as the initial step during biofouling and biofilm formation.51 Herein, the antimicrobial and antifouling efficacies of the pristine


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and polymer-modified GS surfaces were evaluated by using the gram-positive S. aureus and gram-negative E. coli bacteria. LIVE/DEAD two-color fluorescence-based method was used to observe the distribution of adhered bacteria, in which the viable cells appear green and the dead cells appear red. Qualitative analysis of the bacteria adhesion was conducted by comparing the fluorescence microscopy images after 4 h of incubation in the E. coli bacterial suspension (107 cells/mL). In Figure 7, it is observed that the live bacteria on the polymer brushes-functionalized surfaces (Figure 7e, g, i) are significantly lesser than that on the pristine GS surface and the GS-PDA/CTS surface (Figure 7a, c). Notably, after surface grafting of the Arg-Est polymer brushes, a significant reduction in adhered viable cells is observed on the GS-Arg-Est surface (Figure 7g). The hydrolyzed GS-Arg-Est exhibits an enhanced resistance to E. coli bacteria (Figure 7i), indicating that the hydrolysis-induced polymer brush coating has an improved resistance to bacterial adhesion, as well as a higher antifouling efficiency. As a control experiment, the number of adhered E. coli bacteria on the hydrolyzed GS-Arg-Est surface is slightly larger than that on the GS-Arg-Me surface (Figure 7e), indicating a comparable antifouling efficacy of the zwitterionic polymer brush coatings. These phenomena are in good agreement with the results obtained from water contact angle and protein adsorption measurements, suggesting that the polymer brushes polymerized from Arg-Est indeed exhibits antifouling properties, with the anti-adhesion performance obviously improves after hydrolysis.

Subsequently, the antimicrobial properties of the pristine and functionalized surfaces were investigated. The pristine GS surface lacks antimicrobial capability as indicated by the absence of red spots (dead bacteria cells) in Figure 7b. The GS-PDA/CTS surface exhibits an increased antimicrobial activity since fewer red spots are observed. Furthermore, after grafting of the Arg-Est polymer brushes, the GS-Arg-Est surface (Figure 7h) shows a


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significant amount of dead bacteria cells in comparison to the GS-Arg-Me surface (Figure 7f), demonstrating the strong antimicrobial efficiencies. The large amount of dead bacteria can be attributable to the lethal effect of the quaternary ammonium cations associated with the guanidine groups in the Arg-Est polymer brushes, as well as the concomitant reduction in bacterial affinity.40 In comparison, the hydrolyzed GS-Arg-Est surface remarkably reduces the adhered dead bacteria, and only a few noticeable red spots remains (Figure 7j), suggesting the remaining antimicrobial capabilities and the incomplete hydrolysis of ester bonds in ArgEst polymer brushes during the 15-hour hydrolysis reaction. In Figure 8, similar conclusion is also obtained from analysing the fluorescence images of adhered gram-positive bacteria S. aureus on the pristine and polymer-modified GS surfaces.

A more quantitative bacteria assay was conducted by using ImageJ software to count the fractions of adhered bacteria on the pristine and functionalized surfaces (Figure 9).52 For the analysis of antifouling properties of the functionalized surfaces, the pristine GS surface is defined as control with 100% bacterial adhesion, since it attracts the largest amount of adhered bacteria on the surface (Figure 7a, 8a). In contrast, all the other functionalized substrates show lower adhered fractions of both S. aureus and E. coli, as well as significant differences from the adhered fraction on the pristine GS surface. For example, the GSPDA/CTS surface presents a weaker anti-adhesion capability, with adhered fractions of 74% and 62% for live E. coli and S. aureus, respectively. Significantly, the GS-Arg-Me surface shows the lowest extent of bacterial adhesion of 6% and 9%, respectively for E. coli and S. aureus, indicating excellent antifouling properties induced by the assembly of zwitterionic Arg-Me polymer brushes. The GS-Arg-Est surface also exhibits a low bacterial distribution, but a significant difference between the anti-adhesion performance of the GS-Arg-Me and GS-Arg-Est surfaces can be observed. The respective adhered fraction of E. coli and S.


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aureus increases to 31% and 29% compared to that of the GS-Arg-Me surface. After hydrolysis treatment, the hydrolyzed GS-Arg-Est surface exhibits good antifouling performance with 12% adhered E. coli and 13% adhered S. aureus. The antifouling efficacy is slightly lower than that of the zwitterionic GS-Arg-Me surface. However, the bacteriaresistant ability improves considerably over that of the GS-Arg-Me surface.

To quantitatively access the antimicrobial activities, the GS-Arg-Est surface is chosen as control with 100% adhesion, as it is highly effective to eradicate bacteria (Figure 7h, 8h). The pristine GS surface and all other polymer-functionalized surfaces have lower fractions of dead E. coli and S. aureus, implying the weaker antimicrobial capabilities in comparison to the cationic GS-Arg-Est surface. The pristine GS surface has negligible fraction of 3% and 5% of dead E. coli and S. aureus, respectively. The GS-PDA/CTS surface displays some but weak antimicrobial properties, as evidenced by 29% of adhered dead E. coli and 34% of adhered dead S. aureus. After SI-RAFT polymerization of Arg-Me, the antimicrobial efficacy reduces with lower fractions of 11% and 7% for dead E. coli and S. aureus, respectively. The GS-Arg-Est surface exhibits superior antimicrobial properties since there are at least 20 times more dead bacteria compared to that on the pristine GS surface. The excellent antimicrobial performance results from the grafting of cationic Arg-Est polymer brush coating. In addition, the adhered fractions of dead E. coli and S. aureus on the hydrolyzed GS-Arg-Est surface are only 22% and 17%, respectively. The significantly reduced antimicrobial property, as well as the improved fouling-resistant performance, are in good agreement with hydrolysis-triggered surface property transition from cationic (antimicrobial) to zwitterionic (antifouling).

3.4.3 Diatom Attachment Assays


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Amphora coffeaeformis, a member of raphid diatoms, is also employed as microfouler to evaluate the antifouling efficacy of the pristine and polymer-coated surfaces.53 In Figure 10, the pristine GS surface shows the highest concentration of Amphora cells attachment (100% fraction as control). Subsequently, the GS-PDA/CTS surface exhibits a smaller but still substantial dispersion of attached Amphora cells. The attached fraction on the GS-PDA/CTS surface is about 77%, suggesting a poor antifouling efficacy of the surface. After the functionalization of Arg-Me polymer brushes, the attached fraction of microalgal cells is substantially reduced to only 8%, indicating the superior antifouling performance of the zwitterionic polymer brush coating. Meanwhile, a significantly larger number of attached Amphora cells (31%) are observed on the GS-Arg-Est surface, in comparison to that on the GS-Arg-Me surface. In contrast, there is no significant difference between the adhered Amphora fractions of the GS-Arg-Me and the hydrolyzed GS-Arg-Est surfaces. The hydrolyzed GS-Arg-Est surface exhibits an excellent resistance to Amphora attachment, with a small fraction of 11%, implying a comparable antifouling ability of the hydrolysed surface against microorganisms to that of the zwitterionic GS-Arg-Me surface.

3.5 Cytotoxicity assays MTT assays of 3T3 fibroblasts were carried out to evaluate the cytotoxicity of the ‘smart’ coatings. In Figure 11, the pristine GS surface, as well as the surface functionalized with ArgMe brush, present negligible cytotoxicity against 3T3 cells. The cell viability is significantly reduced after grafting of the Arg-Est brushes, as the quaternary ammonium cations in Arg-Est have an adverse effect on the growth of 3T3 fibroblasts. In contrast, the hydrolyzed GS-ArgEst surface shows a lower cytotoxicity and a higher cell viability (94% for 10-day immersion), in comparison to that of the GS-Arg-Est surface (83% for 10-day immersion). Thus, there is no significant difference between the cell viabilities after 3-day and 10-day of co-incubation


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with the functionalized surfaces, indicating the promising biocompatibility of the polymer brush coatings. The MTT assay results are consistent with the antimicrobial and antifouling evaluations. Moreover, all surfaces exhibit high cell viabilities, with the lowest cell viability of 83% relative to that of the control medium (100%), confirming the good biocompatibilities of the as-prepared surface coatings.

3.6 Stability and durability tests To access the stability and durability of the switchable polymer brush coatings on GS surfaces, the polymer-functionalized GS surfaces were exposed to filtered (0.2 µm filter) natural seawater for 30 days. XPS spectral analysis of the aged substrates were conducted. Comparison of the chemical compositions before and after the exposure was used to deduce the stability and durability of the coatings. As shown in Figure S1 (Supporting Information), there are no appreciable changes in the XPS wide-scan spectrum and C 1s core-level spectral lineshape of the aged GS-Arg-Me and aged hydrolyzed GS-Arg-Me surfaces, in comparison to that of the GS-Arg-Me and hydrolyzed GS-Arg-Me surfaces (Figure 2), demonstrating the promising stability and durability of the switchable polymer brush coatings grafted by SIRAFT polymerization.

4. Conclusion Polymer brush coatings with switchable functionalities from antimicrobial (cationic) to antifouling (zwitterionic), were fabricated via SI-RAFT polymerization. Arginine (Arg) and its derivatives were engineered to impart antifouling or antimicrobial properties for the first time. Guanidine-rich Arg-Est polymer brushes were grafted from the GS-PDA/CTS surfaces to prepare the antimicrobial coatings. Hydrolysis of the ester bonds in Arg-Est switches the


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surface functionality, with the property transition from antimicrobial (cationic) state to antifouling (zwitterionic) state. Characterization of surface wettability, surface zeta potential and surface roughness confirmed the “smart coatings” with switchable functionalities. Moreover, the resulting polymer brush-coated GS surfaces exhibited good resistance to the adsorption of nonspecific BSA protein, the adhesion of both gram-positive bacteria S. aureus and gram-negative bacteria E. coli., as well as the attachment of Amphora coffeaeformis. The overall antifouling efficacies of the GS-Arg-Est surface were obviously improved after the hydrolysis treatment, which are comparable to that of the zwitterionic GS-Arg-Me surface. In addition, the switchable coatings were ascertained to be stable, durable and biocompatible. Therefore, the arginine-based “smart coatings” with regulatable functionalities are potentially useful for combating biofouling.

Acknowledgement The authors would like to acknowledge the financial support for this study from Singapore Millennium Foundation under Grant No. 1123004048 (NUS WBS No. R279-000-428-592).

Supporting Information Surface characterization and XPS spectra of the aged GS-Arg-Me and aged hydrolyzed GSArg-Me surfaces (Figure S1). This material is available free of charge via the Internet at http://pubs.acs.org.

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Captions for Scheme and Figures Scheme 1. Schematic illustration of the fabrication of the hydrolysis-triggered antifouling coatings on GS surface via SI-RAFT polymerization. Figure 1. 1H NMR spectra of the synthesized Arg-Me (top) and Arg-Est (bottom) in D2O. Figure 2. XPS wide-scan, C1s and N1s core-level spectra of the (a and b) GS-PDA/CTS, (c and d) GS-Arg-Me, (e and f) GS-Arg-Est, and (g and h) hydrolyzed GS-Arg-Est surfaces. Figure 3. Static water contact angles of the pristine GS, GS-PDA/CTS, GS-Arg-Me, GSArg-Est and hydrolyzed GS-Arg-Est surfaces (Three replicates for each surface and error bar represents standard deviation). Figure 4. Zeta potential of the pristine GS, GS-PDA/CTS, GS-Arg-Me, GS-Arg-Est and hydrolyzed GS-Arg-Est surfaces as a function of pH ranging from 5.0 to 10.0 (Two replicates for each surface and error bar represents standard deviation). Figure 5. AFM Images of the (a) GS-PDA/CTS, (b) GS-Arg-Me, (c) GS-Arg-Est and (d) hydrolyzed GS-Arg-Est surfaces. Rs is the root mean square roughness. Figure 6. SPR sensorgrams of the pristine Au, Au-PDA/CTS, Au-Arg-Me, Au-Arg-Est and hydrolyzed Au-Arg-Est surfaces against the injection of BSA and washing with HEPES. Figure 7. Fluorescence microscopy images of the (a, b) pristine GS, (c, d) GS-PDA/CTS, (e, f) GS-Arg-Me, (g, h) GS-Arg-Est and (i, j) Hydrolyzed GS-Arg-Est surfaces after exposure to E. coli culture (107 cells/mL) at 37 °C for 4 h. Scale bar: 50 µm. Figure 8. Fluorescence microscopy images of the (a, b) pristine GS, (c, d) GS-PDA/CTS, (e, f) GS-Arg-Me, (g, h) GS-Arg-Est and (i, j) Hydrolyzed GS-Arg-Est surfaces after exposure to S. aureus culture (107 cells/mL) at 37 °C for 4 h. Scale bar: 50 µm. Figure 9. Fractions of S. aureus and E. coli adhered on the pristine and functionalized GS surfaces after immersion in the bacterial suspension at 37 °C for 4 h (Three replicates for each surface and error bar represents standard deviation. * denotes significant difference (P

Arginine-Based Polymer Brush Coatings with Hydrolysis-Triggered

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