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A Simultaneously Antimicrobial, Protein-repellent and Cell-compatible Polyzwitterion Network Monika Kurowska, Alice Eickenscheidt, Diana Lorena Guevara-Solarte, Vania T. Widyaya, Franziska Marx, Ali Al-Ahmad, and Karen Lienkamp Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00100 • Publication Date (Web): 07 Mar 2017 Downloaded from http://pubs.acs.org on March 8, 2017

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A Simultaneously Antimicrobial, Protein-repellent and Cell-compatible Polyzwitterion Network Monika Kurowska,1 Alice Eickenscheidt,1 Diana-Lorena Guevara-Solarte,1 Vania T. Widyaya,1 Franziska Marx,1 Ali Al-Ahmad,2 and Karen Lienkamp1,* AUTHOR ADDRESS 1

Bioactive Polymer Synthesis and Surface Engineering Group, Department of Microsystems Engineering (IMTEK) and Freiburg Centre for Interactive Materials and Bioinspired Technologies (FIT), Georges-Köhler-Allee 103, 79110 Freiburg, Germany

2

Department of Operative Dentistry and Periodontology, Center for Dental Medicine of the AlbertLudwigs-Universität, Freiburg, Hugstetter Str. 55, 79106 Germany.

* [email protected]

KEYWORDS Antimicrobial polymer, antibiofouling polymer, biofilm, polynorbornen, polyzwitterion, protein-repellent polymer,

ABSTRACT A simultaneously antimicrobial, protein-repellent and cell-compatible surface-attached polymer network is reported, which reduced the growth of bacterial biofilms on surfaces through its multifunctionality. The coating was made from a poly(oxonorbornene)-based carboxybetaine (PZI), which was surface-attached and cross-linked in one step by simultaneous UV-activated CH insertion and thiol-ene reaction. The process was applicable to both laboratory surfaces like silicon, glass and gold, and real life surfaces like polyurethane foam wound dressings. The chemical structure and physical properties of the

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PZI surface and two reference surfaces (an antimicrobial but protein-adhesive SMAMP polymer coating, and a protein-repellent but not antimicrobial polyzwitterion coating (PSB)), were characterized by FTIR spectroscopy, ellipsometry, contact angle measurements, photoelectron spectroscopy (XPS) and swellability measurements (using surface plasmon resonance spectroscopy, SPR), zeta potential measurements, and atomic force microscopy (AFM). The time-dependent antimicrobial activity assay (time-kill-assay) confirmed the high antimicrobial activity of the PZI; SPR was used to demonstrate that it was also highly protein-repellent. Biofilm formation studies showed that the material effectively reduced the growth of E. coli and S. aureus biofilms. Additionally, it was shown that the PZI was highly compatible with immortalized human mucosal gingiva keratinocytes and human red blood cells using the Alamar Blue assay, the Live-Dead stain, and the hemolysis assay. The PZI thus may be an attractive coating for biomedical applications, particularly for the fight against bacterial biofilms on medical devices and in other applications.

MANUSCRIPT Introduction. Bacterial biofilms that grow on medical products, for example on catheters and wound dressings, cause severe infections that cost the lives of more than hundred thousand people every year worldwide.1-3 Combined with a growing resistance of bacteria like Escherichia coli, Staphylococcus aureus (MRSA), Klebsiella pneumoniae and Enterococcus faecalis against conventional antibiotics, biofilms have become a serious menace to patients even in the most advanced healthcare settings.1,

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Therefore, simple yet

efficient medical coatings that are able to reduce bacterial infections and biofilm formation are urgently needed. In past years, scientists have taken tremendous efforts to develop materials that can stall biofilm formation.5, 6 The key idea behind the design of most of these materials was to interfere with the early

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steps of biofilm formation, as this would most efficiently slow down the biofilm formation rate. The first step in biofilm formation is the adhesion of proteins and other biomolecules to a surface, which happens within seconds.7 When bacteria adhere on this conditioning layer, this is at first reversible, until the bacteria secrete adhesins (sticky proteins) to settle down irreversibly. Bacteria then form colonies and a joint extracellular matrix (ECM) with other pathogens, e.g. fungi like Candida albicans. Inside the ECM, the microorganisms are well protected against antibiotics and the bodies’ own immune system. Eventually, the ECM ruptures due to continuing bacterial proliferation, and planktonic bacteria are released into the organism.8 Thus, the infection is spread, which is the reason why biofilms can be so dangerous. Studies showed that the dose of antibiotics needed to be increased by a factor of up to 1000 to be effective against bacteria in a biofilm.9 At this concentration, however, they may already be toxic to humans. The materials able to interfere with the early stages of biofilm formation can be roughly classified into antimicrobial and “anti-fouling” or protein-repellent coatings.1 Anti-fouling/protein-repellent materials prevent the settling of proteins on surfaces and thus also make bacterial adhesion to the surface more difficult. Antimicrobial coatings, on the other hand, stall or slow down bacterial proliferation. Both fields have been excellently reviewed previously.5, 6, 10 Coatings that impair adhesion of proteins and bacteria again fall into two categories. “Fouling-release” coatings like poly(dimethyl siloxane) have a low mechanical moduli. Proteins and bacteria can adhere to them due to their high hydrophobicity, but they are easily sheared off, e.g. by flowing liquid, from these elastic coatings.6, 10 “Non-fouling” polymers, on the other hand, have such a low interfacial energy that bacterial adhesion on these materials remains reversible.10 For example, poly(ethylene glycol) (PEG), the gold standard for non-fouling coatings, has promisingly low protein adhesion in vitro, but unfortunately can undergo oxidative degradation and chain cleavage and is thus problematic for long-term applications.10 Therefore, other non-fouling coatings, in

1

The term „anti-fouling“ is ill defined. It refers to materials that resist fouling, however it does not specify the nature of the fouling molecule or organism. It may refer to protein-repellent, bacteria-repellent or marine organism-repelling surfaces.

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particular strongly swelling hydrophilic polymers like poly(dimethyl acrylamide),11 have been developed. Polyzwitterions and polyzwitterion-based coatings12, 13 have also been extensively explored13-17 as nonfouling coatings because they mimic the properties of the outer membrane of mammalian cells, which consists of zwitterionic phospholipids. In particular, Jiang and coworkers demonstrated that carboxybetaine-based polyzwitterions reduced long-term biofilm formation (> 100 h) by various bacterial species by 95 % under flow conditions.18 While above mentioned approaches were often very useful against protein and bacterial adhesion in vitro, some of them failed in real-life applications because they were vulnerable to lipid fouling, which enables bacteria to settle on the adhered layer of lipids.19 Additionally, once even single bacteria manage to settle (e.g. on debris, adhered lipids, or minor surface defects), these can form a biofilm in less than 24 hours.20 Coatings with antimicrobial components, on the other hand, kill bacteria before they form biofilms, or at least slow down their proliferation rate. “Leaching” antimicrobial coating consist of an often polymeric carrier matrix that gradually releases an active ingredients (e.g. antibiotics, heavy metals like silver, or other biocides).21 Such materials have been successfully applied in vitro; however there is still no satisfactory proof-of-principle that these materials sufficiently reduce biofilm-formation in clinical settings.22,

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Additionally, these materials lose activity when their active component reaches sublethal

concentrations, which may cause bacterial resistance in the patient or downstream in hospital waste water and eventually the environment. Non-leaching antimicrobial coatings are made from intrinsically antimicrobial cationic polymers.5 These polycationic surface-attached coatings can electrostatically bind bacteria (due to their negatively charged cell envelopes) and damage their membranes by local, physical effects, which are a result of the polymers’ charges and hydrophobic groups.24 After the pioneering work of Tiller and Klibanov in this field,5,

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surface-attached polycationic „Synthetic Mimics of

Antimicrobial Peptides“ (SMAMPs),24, 27-29 which imitate the activity of the natural antimicrobial peptides, were shown to have excellent antimicrobial activity, in some cases combined with low toxicity to human cells, and are thus promising candidates for medical applications.24, 28, 30-34 However, the weak spot of such

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polycationic surfaces it that they collect the negatively charged debris of the killed bacteria, which means that they are exhausted once they are fully covered by bacterial debris. Further attempts to overcome the deficiencies of either antimicrobial or anti-fouling/protein-repellent surfaces included the development of bifunctional materials made from anti-adhesive and antimicrobially active components.6, 35, 36,37-42 However, most of these materials either had limited dual anti-adhesive and antimicrobial activity, or were so complicated to make that they are only of academic interest. Jiang et al. presented fascinating materials that were switchable from antimicrobial to protein-repellent and vice versa,43 a very promising approach once the regeneration of these surfaces can be achieved under more benign conditions. In summary, the field is so far lacking a simple polymer coating with lasting dual antimicrobial and protein-repellent activity to overcome the problems of bacterial biofilms in medical settings and other industries. We here report a polymer coating that could contribute to a solution for this problem. It is based on a carboxybetaine polyzwitterion (PZI, Figure 1), and is to the best of our knowledge the first report of a simultaneously antimicrobial, protein-repellent and cell-compatible polymer consisting of just one active component, and the first report of an antimicrobial polyzwitterion.2 We present an easy way to immobilize this polymer as a surface-attached network on both laboratory and real life surfaces. We further show that this PZI is even more strongly antibacterial than one of the most potent antimicrobial SMAMP polymers, that it significantly reduces biofilm formation, and that it has a low level of toxicity towards human cells. It is thus an extremely attractive coating for biomedical applications, particularly for the fight against bacterial biofilms on medical devices and in other applications.

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Patent pending.

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Figure 1: Structure of the simultaneously antimicrobial and protein-repellent polyzwitterion (PZI) and two reference molecules, the antimicrobial, protein-adhesive SMAMP and the protein-repellent, nonantimicrobial PSB.

PZI

SMAMP

PSB

Experimental General. All chemicals were obtained as reagent grade from Sigma-Aldrich or Carl Roth and used as received unless otherwise indicated. Dichloromethane (DCM) was distilled from CaH2 under nitrogen gas. Gelpermeation chromatography (GPC) was measured in chloroform or tetrahydrofuran (THF) on a PSS SDV column (PSS, Mainz, Germany) and calibrated with PMMA standards. Solvents for GPC were HPLC quality and obtained from Carl Roth. NMR spectra were recorded on a Bruker 250 MHz spectrometer (Bruker, Madison, WI, USA). Electron ionization mass spectra were measured on a Thermo TSQ 700 spectrometer (Thermo Scientific, ionization energy 70eV, source temperature 150°C). Optical and fluorescence microscopy images were taken on a Nikon Eclipse Ti-S inverted microscope (Nikon GmbH, Düsseldorf, Germany). Synthesis. Synthesis of molecules for surface attachment. The molecules used for surface attachment of the PZI networks were 3EBP (for all surfaces except gold), and LS-BP (for gold surfaces used in surface plasmon

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resonance spectrscopy (SPR) experiments). LS-BP and 3EBP-silane were synthesized as described in the literature.39, 44 Synthesis of the zwitterion precursor monomer. The zwitterion precursor monomer was obtained from exo-7-oxabicyclo[2.2.1]hept-5-ene-2,3-dicarboxylic acid anhydride (5 g, 30.0 mmol, 1 eq.), which was dissolved in CH2Cl2. 1.1 eq of N-(tert-butoxycarbonyl)ethanolamine (5.32 g, 33 mmol, 1.1. eq.) and 10 mol% 4-dimethylaminopyridine (DMAP) were added. After stirring over night, the solution was washed with 10% KHSO4 and water, and dried over Na2SO4. The solvent was removed by evaporation under reduced pressure and the product was dried under high vacuum. A white solid was obtained. The isolated yield was 70%. 1H-NMR (250 MHz, CDCl3): 1.46 (s, 9H,CH3), 2.87 (m, 2H, -(-C=O)O-CH), 3.38 (m, 2H, N-CH2), 4.03-4.31 (m, 2H, O-CH2), 5.80-5.05 (br s, 1H, NH), 5.26 & 5.34 (m, 2H, bridge head H), 6.47 (m, 2H, H-C=C); 13C-NMR (62.9 MHz, CDCl3): 28.35 (CH3), 39.60 (N-CH2) -O-C-CH3), 46.78 & 47.35 (-(C=O)-CH), 64.44 (O-CH2), 80.08 & 80.52 (bridge head C), 136.41 & 136.58 (C=C), 171.44 & 174.59 (C=O); MS: m/z = 328.14 (M+ ̇ ), 283.09 (M-COOH), 272.07 (M-tBu), 226.03 (M-CO2tBu). Polymerization of the zwitterion precursor monomer. The polymerization of the zwitterion percursor monomer was performed under nitrogen using standard Schlenk techniques. The zwitterion monomer precursor (500 mg, 1.2 mmol) was dissolved in 5 mL tetrahydrofuran (THF). The Grubbs third generation catalyst G3 (3.7 mg, 5 µmol) was dissolved in 2 mL dry DCM and added in one shot to the vigorously stirring monomer solution at room temperature. After 30 min, the living polymer chain was end-capped with an excess of ethylvinyl ether (1 mL, 750 mg, 10 mmol). The solution was allowed to stir for 1 hour. The solvent was then evaporated under reduced pressure. The product was re-dissolved in a small amount of ethyl acetate and added dropwise into ice-cooled n-hexane. The colorless precipitate was removed by filtration, and dried in dynamic vacuum. The isolated yield was 90%. For GPC measurement, the polymer was derivatized with trimethylsilyldiazomethane according to a literature procedure.[2] GPC (THF, r.t., 1 mL min-1): Mn = 70,000 g mol-1, Mw = 111 000 g mol-1, PDI = 1.5; 1H-NMR (250 MHz, THF-d8): 1.41 (s,

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9H, CH3), 3.10 (br m, 2H, -(C=O)-C-H), 3.28 (br m, 2H, N-CH2), 4.08 (br m, 2H, O-CH2), 4.67 (m, 1H, C=C-CH trans), 5.10 (br s, 1 H, C=C-CH cis), 5.59 (br s, 1H, NH), 5.88 (br m, 1H, C=CH cis) and 6.08 (br m, 1H, C=CH trans); 13C-NMR (62.9 MHz, THF-d8): 28.93 (CH3), 40.37 (N-CH2), 53.83 & 54.40 ((C=O)-C-H), 64.98 ((-O-C-CH2), 78.93 (C-CH3), 81.44 & 81.88 (C=C-CH), 133.42 & 133.60 (C=C), 156.86 (N-C=O) 171.36 & 172.72 (C=O). Polyzwitterion precursor polymer deprotetction. The N-Boc protected zwitterionic polymer (500 mg) was dissolved in 20 mL of dry THF under nitrogen. To this solution, 20 mL of 4 M HCl in dioxane was added. After a few minutes, 10 vol% methanol were added to maintain solubility of the hydrolyzing polymer. The mixture was stirred over night at room temperature. The solvent was then evaporated under reduced pressure. The crude polymer was re-dissolved in methanol and precipitate into ice-cooled diethyl ether nhexanes (90:10 vol%) mixture. 1H-NMR (250 MHz, MeOH-d4): 3.35 (br s, 2H, (C=O)-CH + solvent), 3.72 (br s, 2H, N-CH2), 4,40 (br s, 2H, O-CH2), 4.74 (m, 1H, C=C-CH + solvent), 5.16 (br s, 1 H, C=CCH cis), 5.73 (br s, 1H, C=CH cis) and 5.97 (br s, 1H, C=CH trans); 13C-NMR (62.9 MHz, MeOH-d4): 40.10 (N-CH2), 53.42 & 53.99 ((C=O)-CH), 62.76 (O-CH2), 78.63 & 82.43 (C=C-CH), 132.66 & 133.65 (C=C), 173.06 & 172.28 (C=O). Synthesis of butyl SMAMP monomer. The butyl SMAMP monomer was synthesized and characterized as previously published.28, 45 Polymerization of butyl SMAMP monomer. The polymerization of the butyl SMAMP monomer was performed under nitrogen using standard Schlenk techniques. The Butyl monomer (500 mg, 1.42 mmol) was dissolved in 3 mL dry DCM. The G3 catalyst (0.72 mg, 1.1 µmol) was dissolved in 1 mL dry DCM in a second flask and added in one shot to the vigorously stirring monomer solution at room temperature under N2. After 30 min, excess ethylvinyl ether (1 mL, 750 mg, 10 mmol ) was added. The mixture was stirred for 1 hour. The solvent was then evaporated under reduced pressure. The product was re-dissolved in a small amount of DCM and added dropwise into ice-cooled n-hexane. The colorless precipitate was

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removed by filtration, and dried in dynamic vacuum. The NMR signals of the polymer matched those in the literature.46 GPC analysis (Chloroform, r.t., 1 mL min-1): Mn = 236,000 g mol-1, Mw = 260,000 g mol-1, PDI = 1.1; 1H-NMR and 13C-NMR data matched the literature values.28 Synthesis of poly(sulfobetaine) (PSB) monomer. The PSB monomer was synthesized and characterized as previously published.47 Polymerization of poly(sulfobetaine) (PSB) monomer. The polymerization of the PSB monomer was performed under nitrogen using standard Schlenk techniques. Monomer (500 mg, 1.34 mmol) was dissolved in 5 ml 2,2,2-trifluoroethanol (TFE). In another Schlenk flask, G3 catalyst (7.27 mg, 0.01 mmol) was dissolved in 2 ml dry DCM. After dissolving both solids, the monomer solution was degassed by three-pump-thaw cycles. After the solution came to room temperature, the monomer solution was added in one shot via syringe to the catalyst solution. The reaction mixture was stirred for 30 min at room temperature. The polymerization was terminated by addition of excess ethylvinyl ether (1 ml, 750 mg, 10 mmol) and stirred for 1 hour. The solvent was removed under reduced pressure. The product was redissolved in a small amount of TFE and precipitated into ice-cooled diethyl ether. The NMR signals of the polymer matched those in the literature.47 Surface functionalization. Functionalization of model surfaces with surface functionalization agents. Silicon wafers: A solution of 3EBP-silane (20 mg mL−1 in toluene) was spin coated on a 525 ± 25 µm thick, one-side-polished 100 mm standard Si (CZ) wafer ([100] orientation) at 1000 rpm for 120 s. The wafer was cured for 30 min at 120°C on a preheated hot plate, washed with toluene and dried under a continuous nitrogen flow. Glass substrates were functionalized analogously.

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Gold substrates: For SPR measurements, the LaSFN9 glass slides coated with a 1 nm chromium layer (adhesive layer for gold) and a 50 nm gold layer were covered with a 5 mM solution of LS-BP in toluene for 24 h. Then, the samples were washed with toluene and ethanol, and dried under nitrogen flow. SPR measurements indicated that the thickness of the LS-BP layer was 1 nm. Wound dressing: Commercially available polyurethane-based wound dressing foam (Suprasorb P, Lohmann und Rauscher, Rengsdorf, Germany, 5 mm thickness) was cut into 2 x 2 cm pieces, which were immersed into a 5 mM solution of LS-BP in toluene for 24 h. Then, the samples were washed with toluene and ethanol, and dried under nitrogen flow. Immobilization of polymer networks on silicon wafers, gold and glass substrates functionalized with benzophenone: Polyzwitterion (PZI) network. (a) From PZI precursor polymer with Boc protective group: A stock solution (Solution A) was prepared by dissolving pentaerythritol-tetrakis-(3-mercaptopropionate) (1 mL, 1.3 g, 2.6 mmol) in THF (50 mL). The polyzwitterion precursor polymer (10 mg, 0.027 mmol) was dissolved in Solution A (0.25 mL). Chloroform (0.4 mL for silicon coating; 0.8 mL for gold or glass coating) was added as co-solvent. The mixture was stirred for 60 s. From this solution, a polymer film was spin cast on a 3-EBP treated silicon wafer, a LS-BP treated gold substrate, or a 3-EBP treated glass substrate at 3000 rpm for 30 s. The resulting polymer film was cross-linked at 254 nm for 30 min in a BIO-LINK Box (Vilber Lourmat GmbH, Eberhardtzell, Germany). It was then washed with THF to remove unattached polymer chains and dried under N2-flow. This yielded the PZI precursor network. To remove the Boc protective groups, the film was immersed in HCl (4 M in dioxane) for 12 hours and washed twice with ethanol. It was then dried under N2-flow to yield the PZI network. (b) From deprotected PZI: A stock solution of cross-linker was prepared by dissolving pentaerythritol-tetrakis-(3mercaptopropionate) (0.1 mL, 0.13 g, 0.26 mmol) in ethyl acetate (5 mL). The deprotected PZI was dissolved in 0.8 mL methanol. Then 0.2 mL cross-linker solution in ethyl acetate was added and the

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mixture was stirred for 60 s. From this solution, a polymer film was spin cast direct on a silicon wafer at 3000 rpm for 30 s. The film was cross-linked at 254 nm for 30 min in a BIO-LINK box. It was then washed with methanol to remove unattached polymer chains and dried under N2-flow. Butyl SMAMP network. A stock solution (Solution B) was prepared by dissolving pentaerythritol-tetrakis(3-mercaptopropionate) (1 mL, 1.3 g, 2.6 mmol) in dichlormethane (50 mL). The Butyl SMAMP precursor polymer (10 mg) was dissolved in Solution B (0.25 mL). Toluene (0.3 mL) was added as cosolvent, and the mixture was stirred for 60 s. The remaining steps were exactly the same as described above for the poly(zwitterion) network. Poly(sulfobetaine) (PSB) network. A stock solution (Solution C) was prepared by dissolving pentaerythritol-tetrakis-(3-mercaptopropionate) (0.1 mL, 0.13 g, 0.26 mmol) in 2,2,2-trifluoroethanol TFE (5 mL). The PSB polymer (30 mg) was dissolved in Solution C (0.25 mL). TFE (0.8 mL) was added to adjust the desired coating thickness. The mixture was stirred for 60 s. The solution was spin coated on a 3-EBP treated silicon wafer at 3000 rpm for 10 s. The film was cross-linked at 254 nm for 30 min in a BIO-LINK box. It was then washed with TFE to remove unattached polymer chains and dried under N2flow.

Polymer network characterization. Ellipsometry. The thickness of the dry polymer layers on silicon wafers was measured with the autonulling imaging ellipsometer Nanofilm EP3 (Nanofilm Technologie GmbH, Göttingen, Germany), which was equipped with a 532 nm solid-state laser. A refractive index of 1.5 was used for all measurements. For each sample, the average value from three different positions was taken. Attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). Double side polished silicon wafers were used as substrates for the FTIR experiments. The polymer layer was immobilized on

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one side of a double side polished silicon wafer. The spectra were recorded from 4000 to 400 cm-1 with a Bio-Rad Excalibur spectrometer (Bio-Rad, München, Germany), using a spectrum of the blank double side polished silicon wafer as background. The spectra for PZI, PSB and SMAMP are shown in the supporting information (Table S1). Contact Angle. The contact angle system OCA 20 (Dataphysics GmbH, Filderstadt, Germany) was used to measure the static, advancing and receding contact angles of the SMAMP precursors and the activated SMAMP networks. The average value of the contact angle was obtained from four measurements on different positions of one sample. The static contact angles were calculated with the Laplace-Young method, while the advancing and receding contact angles were calculated with elliptical and tangent methods. The results are summarized in Table S2. Atomic force microscopy (AFM): The topography of the surfaces was imaged with a Dimension FastScan and Icon from Bruker. Commercial FastScan-A cantilevers (length: 27 µm; width: 33 µm; spring constant: 18 Nm-1; resonance frequency: 1400 kHz) and ScanAsyst Air cantilevers (length: 115 µm; width: 25 µm; spring constant: 0.4 Nm-1; resonance frequency: 70 kHz) were used. All AFM images were recorded in tapping mode in air and ScanAsyst in air, respectively. The obtained images were analyzed and processed with the software ‘Nanoscope Analysis 9.1’. For each sample, the root mean square (RMS) average roughness from three images of an area of 5x5 µm² at different positions was taken. The results are summarized in Table S3 in the supporting information. Photoelectron spectroscopy (XPS). XPS Spectra were measured on a Perkin Elmer PHI 5600 ESCA System (PerkinElmer, Waltham, MA, USA). The X-ray source was a Mg anode with an energy of 1253.6 eV. The aperture size was 400 µm, the angle was 45°. The typical measurement size is 10 µm2. Samples were mesured at room temperature. XPS data are shown in Figure 3 and Figure S2 in the supporting information.

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Zeta potential measurements. The streaming current measurements for electrokinetic surface characterization were performed with an electrokinetic analyzer with integrated titration unit (SurPASS, Anton Paar GmbH, Austria). The analyzer was equipped with an adjustable gap cell. Ag/AgCl electrodes were used to detect the streaming current. The respective polymers were spin-cast on fused silica substrates (MaTeC, 20 x 10 x 1 mm lp, Ch.Nr. 13112704) and put into the measuring cell. Before each measurement, the electrolyte hoses were rinsed with ultrapure water until a conductivity of < 0.06 mS m-1 was reached. The measuring cell was mounted and the electrolyte solution (1 mM KCl) was prepared. The pH of the electrolyte solution was adjusted to pH 3.5 with 0.1 M HCl prior to filling the electrolyte hoses. The gap height was adjusted to approx. 105 µm while the system was rinsed for 3 min at 300 mbar. Titration measurement was performed with 0.1 M NaOH. The target pressure of the pressure ramp was set to 400 mbar. After titration and before each measurement cycle, the system was rinsed for 3 min at 300 mbar. The pressure program was: target pressure 400 mbar; max. time 20 s; current measurement; 2 repetitions. The rinse program was: max. pressure 300 mbar; max. time 180 s. The parameters for the pH titration were: pH difference = 0.2; volume increment 0.01 mL; pH minimum 2.5; pH maximum 10.5. Representative titration curves (zeta potential vs. pH) are shown in Figure S1. The data is discussed in the main article. It was fitted with the Hill equation, a standard fit for sigmoidal curve shapes. Substituting the

variables for this fit into the Hill equation, it reads ζ = ζ + (ζ − ζ ) . The fitting parameters were: ζ , the extrapolated start value of the fitted positive plateau; ζ , the extrapolated end value of the fitted negative plateau; k, the point of inflection and n, a measure of the width and steepness of the sigmoidal curve. We also determined the isoelectric point (IEP) from the curve and calculated the acid constant ( value) of the surface bound acid-base pair of the polymer from =

ζ + 0.4343

ζ !

, where ζ is the pH value where the half maximum (or half

minimum) of ζ is measured. The second term (containing Faraday’s constant, the universal gas constant and the absolute temperature) is a term that corrects for the ionic strength.48 We also used the Hill

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equation to calculate ζ under pseudo-physiological conditions by setting pH = 7.4. This is not exactly correct, because ζ is sensitive to ionic strength and was measured at 0.001 mol/L in KCl (= 0.002 osmol L1

), and not under physiological conditions of about 0.3 osmol L-1. However, this high salt concentration

could not be used because it is outside the range in which the ζ can be measured with our set-up. ζ typically decreases with increasing ionic strength. Nevertheless, this value gives an estimate of the surface charge at neutral pH, which is useful when interpreting the biological data. Surface plasmon resonance spectroscopy (SPR) experiments. Setup and sample preparation. SPR measurements were performed on a RT2005 RES-TEC device in Kretschmann configuration from ResTec, Framersheim, Germany. Excitation was done with a He-Ne-Laser with λ= 632.8 nm. SPR substrates were homemade (LaSFN9 glass from Hellma GmbH, Müllheim, Germany; coated with 1 nm Cr and 50 nm Au at the Clean Room Service-Center (RSC) of the Department of Microsystems Engineering, University of Freiburg, using the device CS 730 S (Von Ardenne, Dresden, Germany). SPR angular reflectivity scans and determination of the swelling ratio of the polymer networks. To study the swelling of the polymer networks, full reflectivity curves (reflectivity vs. angle of incidence) were measured against air and water. The thickness of layer of the material was calculated by simulations of the reflectivity curves based on the Fresnel equations, which were performed with the software ‘Winspall’. The following permittivities ε’ and ε’’ were used: LaSFN9 (ε’ = 3.4036; ε’’ = 0); Cr (ε’ = –6.3; ε’’ = 20); Au (ε’ = –12.3; ε’’ = 1.3), PZI (ε’ = 2.43; ε’’ = 0), PSB (ε’ = 2.25; ε’’ = 0), SMAMP (ε’ = 2.5; ε’’ = 0), fibrinogen (ε’ = 2.25; ε’’ = 0), nitrogen (ε’ = 1; ε’’ = 0). For the swelling experiments, relatively thick samples (at least 200 nm or more) were used, which would not only give rise to a plasmon peak in the reflectivity vs. angle curve, but also to waveguide peaks. This allows for more precise data fitting. In each swelling experiment, the SPR reflectivity curve of dry polymer network was recorded first. Then, solvent was injected into the flow cell, and after equilibration for at least 30 minutes, the SPR reflectivity curve of the swollen material was recorded. After simulation, the sample thickness d and the real part of the

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permittivity (ε’) of the polymer network were obtained by fitting the calculated curve to match the minimum of the wave guide modes. The swelling ratio of the polymer network was calculated by " = #$%&'( ) . #*+,

SPR curves of the swollen and non-swollen material are shown in Figure S5.

SPR kinetics experiments. Protein adsorption was studied in the kinetics mode. For this set of experiments, thinner layers (see Table S6) giving only rise to plasmon signals but not to waveguides were used. To set up the experiment, an angular scan of the substrate under HEPES flow was performed to detect the plasmon signal minimum. The protein adsorption experiments in the kinetic mode were then carried out at -. = - − 1 (on the left flank of the plasmon peak), by monitoring signal intensity at that angle vs. time. First, the baseline intensity at that angle against buffer was recorded for about 20 min. Then, the protein solution was injected (Fibrinogen solution at a concentration of 1 mg ml-1, in HEPES buffer, flow rate 50 µl h-1; the first arrow in the kinetics figures marks the time point of protein injection). After about 20-30 min, protein adsorption would reach an equilibrium (plateau in the curve). At this point (second arrow in the kinetics figures), buffer was injected to remove any loosely adhering protein. The difference of the reflectivity value before protein injection and after the final buffer wash gives a qualitative estimate whether the surface is protein-adhesive, or not. To quantitatively determine the average thickness of adsorbed fibrinogen layer after the kinetics experiment, the surfaces were rinsed with MilliQ water for 15 min to remove residual salt, and dried under nitrogen flow. Afterwards, another angular reflectivity curve was measured. The thickness of each layer of the material was calculated by simulations of the reflectivity curves based on the Fresnel equations, as described above. The covering density of the adsorbed protein on the surface was calculated by: Γ =

0

= 0∙∆# ∙

0∙∆# 0

= 3 ∙ ∆4, where Γ is the average protein layer

thickness (in g mm-2), m is mass of the adsorbed protein, A is the surface area, ∆d is the protein layer thickness, and ρ is the density of the protein. The literature value for the density of the fibrionogen is 1.085 g cm-3.[5]

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Bioactivity Studies. Time-dependent antimicrobial activity assay (time-kill-assay). Experiments to test the antimicrobial activity of the polymer networks were performed using a modification of the Japanese Industrial Standard JIS Z 2801:2000 ‘Antibacterial Products Test for Anti-bacterial Activity and Efficacy’ as reported previously.49, 50 S. aureus (ATCC29523) and E. coli (ATCC25922) were cultured overnight in triptic soy broth and diluted 1:10. Optical density (OD) was checked 3-4 hours later and the bacterial culture (1.5 ml of S. aureus and 150 µL of E. coli) was mixed in a chromatography sprayer bottle with 100 ml of sterile NaCl 0.9 % solution and continuously stirred.49 The test samples (5 of each material), and a positive and a negative control, were each fixed at the center of sterile Petri dishes and placed at a distance of 15 cm to the spray nozzle of the spray bottle. The bacterial suspension (concentration of about 106 bacteria per cm3 according to OD reading) was sprayed onto the samples using compressed air from a 50 mL syringe.24 Afterwards, the petri dishes were immediately covered and incubated for 2 h in a humid chamber at 37°C under aerobic conditions and 5 % CO2. A small drop (50 µL) of sterile 0.9 % NaCl solution was added onto a defined area of the samples and left for 2 min. It was then sucked from the surface with an Eppendorf pipette. To ensure removal of bacteria from the surface the solution was pumped back and re-pipetted twice and finally spread over Columbia blood agar plates. These were incubated overnight at 37°C without agitation to allow any surviving bacteria to form colonies. These colony forming units (CFUs) were counted using the software ‘Quantitiy One’. Each experiment was performed at least twice. The CFUs were reported as % Growth relative to the negative and positive control. The negative control (growth control) was an uncoated silicon wafer piece, the positive control was a silicon wafer that had been incubated with chlorohexidine digluconate (CHX). The Growth $?&( @ ?%$. A% )+%&

percentage was calculated as % 6789:ℎ =

(B. A% )+%& @ ?%$. A% )+%&

∙ 100. The data obtained is

shown in Figure 5. Biofilm formation studies. The test samples were silicon wafer pieces with a size of 5 x 5 mm were cut from 15 x 15 mm pieces coated with the different polymer networks. The positive control and the negative

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control were uncoated silicon wafer pieces. The samples and control pieces were placed into a sterile 24– well microplate using sterilized tweezers. 1000 µL of bacterial overnight culture in tryptic soy broth (TSB) medium was added to each well. The bacteria tested in each experiment were S. aureus ATCC29523 and E. coli (ATCC25922). All the samples sets were incubated at 37°C without agitation and 5 % CO2 for 2 h, then 5000 µL of TSB (enriched with sucrose) was added to each well, and incubated for different times (12 h, 24 h, 48 h, 72 h) under these conditions. After these incubation times, the samples were placed in a microplate and all of them were washed 3 time with NaCl in order to remove the nonadherent microorganisms. Each well was stained using the Life/Dead stain (Live/Dead BacLight bacterial viability kit, Molecular Probes, Eugene, OR, USA) according to the instructions of the manufacturer. The samples were stored for 10 min in a dark chamber. After that, every sample was placed face down in an Ibidi µ-Slide 8 well chamber. Images (red and green channel) were recoreded using a fluorescence microscope (Zeiss Axio Observer.Z1, with an objective of 63x oil). The excitation/emission maxima for the dyes were 500 nm for the SYTO 9 stain (green-fluorescent, “Live”) stain and 617 nm for the propidium iodide stain (red-fluorescent, “Dead”). The objective was covered with immersion oil (Zeiss inmersol). The results thus obtained for S. aureus and E. coli are shown in Figure S2 and S3, respectively (overlay of green and red channel). Additionally, the green flurescence intensity of three representative positions were evaluated for each sample using the Software Zen 2 (blue edition). The resulting data, normalized to the growth control (NC), is shown in Figure 6, some representative micrographs are shown in Figure S3 and S4 of the supporting information. Cell assays. Ethics statement. Gingival mucosal keratinocyte and red blood cells were obtained from human volunteers who had previously given their written consent according to the Helsinki declaration. This was approved by the ethics vote number 381/15 of the Ethics Board of the Albert-Ludwigs University, Freiburg, Germany.

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Optical microscopy and Alamar Blue Assay. Alamar Blue assay experiments were performed on round glass microscope coverslips (22 mm diameter, thickness 0.5 mm; Langenbrick, Emmendingen, Germany), which had been coated with the test polymer networks as described above. Coverslips without coating were used as controls. Before starting the experiment, coverslips used as controls were washed 30 minutes in 100% isopropanol to emulate the process used when coating the test samples. Thereafter, both control and sample coverslips were sterilized for 15 minutes in 70% ethanol. All coverslips (test samples and controls) were subsequently washed 3 times with PBS buffer in order to remove residual ethanol. Coverslips (samples and controls) were tested in triplicate and placed in 12-well plates (bio-one Cellstar, Greiner, Frickenhausen, Germany). Cell treatment: Immortalised HPV-16 gingival mucosal keratinocyte (GM-K) cells were cultivated in keratinocyte growth medium (KGM) (Promocell, Heidelberg, Germany) with accompanying supplements prepared at concentrations supplied by the manufacturer: bovine pituitary extract – 0.004 mg mL-1; epidermal growth factor (EGF) – 0.125 ng mL-1; insulin – 5 µg mL-1; hydrocortison – 0.33 µg mL-1; epinephrine – 0.39 µg mL-1; transferin - 10 µg mL-1; CaCl2 – 0.06 mM; in addtion to the antibiotic kanamycin at 100 µg mL-1. Cells were trypsinised at between 70–90 % confluency and resuspended in supplement / antibiotic free KGM. They were then seeded out onto test and control surfaces in 1 mL medium at 1.5 x 105 cells mL-1 in supplement / antibiotic free medium. Thereafter, the 12 well plates containing the cells were incubated at 37°C/ 5 % CO2 for 5 hours, allowing cells to settle and begin adhesion. After this time, 500 µL of medium from above the cells was carefully aspirated and replaced by 500 µL medium containing double normal supplement concentration, yielding a normal supplement concentration medium. Cells on test surfaces and controls were cultivated for 18 more hours (total 24 hours), 42 h (total 48 h) and 66 h (total 72 h), respectively. At each time point, positive control samples were generated by aspirating 500 µL medium from 3 wells and adding 500 µL 60 % isopropanol to give a 30 % isopropanol solution. A negative control was generated by removing the old medium and replacing it with 1 mL fresh medium. Optical micrographs of the keratinocytes grown on PZI, PSB and the untreated glass slides (growth control) were taken using a phase contrast objective (10x

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magnification) on a Leica DMIL microscope with a Leica D-LUX-3 CCD camera (10x magnification). The results are shown in Figure S5. For the Alamar blue assay, all samples and controls were cultivated for another 30 minutes, after which 110 µL pre-warmed (37°C) Alamar Blue (AbD Serotec, Oxford, UK) was slowly pipetted into each well (samples and controls) with gentle agitation to ensure homogeneous dispersion, giving a 10 % solution. Cells were incubated again for 2 hours, after which time medium from all wells containing Alamar Blue was aspirated and collected into 1.5 mL Eppendorf tubes (one for each well). Tubes were then centrifuged at 1,000 g for 5 minutes to precipitate cells, after which the fluorescence intensity of the supernatant was measured (excitation at 540 nm; measurement at 590 nm) on a Tecan Infinte 200 plate reader. The data was analysed according to the Alamar Blue manufacturer’s instructions. The experimental procedure was repeated at 48 hours and 72 hours to give time dependent data. The results thus obtained (percentage of dye reduction relative to the initial dye concentration) for kerationcytes grown on PZI, SMAMP and PSB for 24, 48, and 72 h are shown in Figure S5. Normalized, relative data is shown in Figure 7 of the main text. Live-Dead staining of keratinocytes grown on polymer networks. Immediately after the supernatant for the Alamar blue assay was removed (72 h time point), the gingiva mucosa keratinocytes (GM-Keratinocytes immortalized with HPV-16) were washed twice with PBS. For live cell staining, Syto16 green fluorescent nucleic acid stain (Molecular Probes, Eugene, OR, USA), diluted 1:200 in keratinocyte growth medium (Promo Cell, Heidelberg, Germany) was used. Propidium iodide (Sigma-Aldrich GmbH, Steinheim, Germany) was used for dead cell staining (dilution 1:1000). Cells were incubated with the stains at 37°C in humidified air containing 5% CO2 for 30 min and washed twice with PBS. Cell viability was determined in PBS immediately afterwards. The samples were examined using the Keyence BZ-9000E flurescence microscope. The images were captured using the software BZ II Viewer and BZ II Analyser. Green fluorescence was measured at ca. 490 nm excitation, red at 536 nm. The image contrast was adjusted to better visualise the staining. Representative pictures of each sample are shown in Figure 8 of the main text.

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Hemolysis Assays. Hemolysis experiments were performed according as previously, with minor modifications.51 In short, EDTA-blood was drawn from a human volunteer and washed with Tris-buffered saline (TBS, pH 7.0, 0.01 M Tris-HCl, 0.155 M NaCl) by adding 30 µL blood to 10 mL TBS in a 15 mL Greiner tube, shaking well by hand, and then centrifuging at 3000 rpm (about 1,000 g) for 5 minutes. The supernatant above the concentrated red blood cells (RBCs) was discarded and a further 10 mL TBS added. This procedure was repeated 3 times to yield plasma free RBCs. To control the cell number and quality, cells were counted before every experiment using the Neubauer chamber and cells were used in a range of >2000 erytrocytes µL-1. Finally, 0.3 % (v/v) RBCs in TBS were used for each experiment. The PZI sample was prepared as stock solution in dimethyl sulfoxide (DMSO) at 40 mg mL-1. Serial pre-dilutions with 10 % DMSO in TBS were made in 1.5 mL tubes (Eppendorf, Hamburg, Germany) in a total volume of 100 µL so that concentrations were stepwise halved 11 times. 40µL of the stock solution was pipetted directly in duplicates on a 96 well plate (Grainer Bio One, Cellstar, Frickenhausen, Germany) to give an initial working solution of 8000 µg mL-1. Additionally, 40 µL of each dilution was pipetted in duplicates to give a total range from 8000 µg mL-1 to 3.9 µg mL-1. 40 µL Melittin from honey bee venom (SigmaAldrich, St. Louis, USA) was used as positive control at a concentration of 80 µg mL-1 (diluted in TBS). 40 µL TBS were used as control (blank) and 100 µL TBS with 100 µL DMSO as background control. Finally, 160 µL of the 0.3 % RBC solution was added to each well containing 40 µL sample or control (except background control). The plate was shaken gently for 1 minute, and then incubated for 3 min at 37°C. Thereafter, the plate was centrifuged at 3000 rpm for 5 minutes. 100 µL of the supernatant was pipetted into a new 96 well plate and the optical density (OD) of each well was measured at 414 nm on a UV-vis plate reader (Tecan infinite M200) to determine the amount of lysed red blood cells. For statistical analysis, blank OD414 values were subtracted from both sample OD414 and positive control OD414 values. OD414 was normalised to 100 % with the lowest OD414 assigned to 0 % hemolysis, and the melittin positive control assigned to 100 % hemolysis. The data was plotted as percentage hemolysis vs. log10 of

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concentration. From this curve, the HC50 value was interpolated. The value derived from 3 independent hemolysis experiments is reported. For PZI, that value was 8000 ± 500 µg mL-1.

Results. Polyzwitterion synthesis and surface coating process. The title material, a carboxybetain-based polyzwitterion (PZI, 1), was synthesized in three steps (Scheme 1). First, monomer 2 was obtained by base-catalyzed ring-opening of the commercially available oxonorbornene-anhydride 3 with N-Bocethanolamine 4. This monomer carries an acid group and a Boc-protected primary amine group. After careful removal of the dimethylaminopyridine (DMAP) auxiliary base, monomer 2 was polymerized by ring-opening metathesis polymerization (ROMP) using Grubbs IIIrd generation catalyst (G3). This yielded the PZI-precursor polymer 5. The Boc protective group was removed from polymer 5 by hydrochloric acid, giving the desired polyzwitterionic PZI 1.

Scheme 1: PZI synthesis. The monomer precursor 2 was obtained in one step from the anhydride 3 and NBoc-protected ethanol amine 4. Polymerization by ROMP with Grubbs 3rd generation catalyst (G3), and deprotection with HCl yielded the PZI 1. 4 O

H N

HO

O

O O

O

O

O

DMAP

3

O

2

H N

O OH

O O

O

G3

Ph

Ph O

O

n

n 3

HCl O

O

O

–

1

NH 3+

O OH O

O O

5

NH O O

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The target polymer was synthesized via the protective group approach described above because polyzwitterions are often poorly soluble, and thus difficult to handle, in consequence of the “antipolyelectrolyte effect”:13 when the positive and negative charges on a polyzwitterionic repeat unit form a tight ion pair, polyzwitterions become insoluble in water and most organic solvents. Addition of salt breaks these ion pairs and improves the solubility of polyzwitterions, but high-salt conditions are undesirable for the formation of coatings. It is probably due to the anti-polyelectrolyte that the so far reported library of synthetic polyzwitterions, and even more so of polyzwitterionic coatings, is rather limited. In the case of the here reported PZI, the N-Boc protected precursor polymer 5 was readily soluble in several organic solvents including dichloromethane and tetrahydrofuran (THF). Thus, polymer characterization was relatively straight-forward. NMR spectroscopy data (included in the Experimental) confirmed the assumed polymer structure, and gel permeation chromatography (in THF) showed that relatively high molecular weights (73,000 g mol-1), with a polydispersity index of 1.5 g mol-1, were obtained. Advantageously, it was also found that the PZI 1 was readily soluble in methanol and thus could be easily handled experimentally. PZI coated surfaces (6) were obtained by applying a solution of the PZI 1 (or its N-Boc protected analogue 5) and the tetrafunctional thiol cross-linker 7 to a benzophenone-functionalized substrate (8) by spin-coating or dip-coating (Scheme 2). The thus coated material was then UV irradiated. The UV irradiation simultaneously triggered two reactions: a CH-insertion reaction between the keto groups of the benzophenone-pretreated substrate44 8 and aliphatic CH groups of the polymer, and a thiol-ene reaction between the polymer double bonds and the thiol groups of cross-linker 7 (Scheme 2). The surface was then washed to remove unbound polymer chains. Importantly, either the deprotected PZI 1 (in methanol) or the precursor polymer 5 (in THF) could be used to coat the target surface. In the latter case, the surface was treated with HCl to remove the Boc-protective groups, so that the desired PZI coating 6 was obtained.

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The networks synthesized for the physical characterization of the PZI on standard laboratory surfaces like glass, quartz, silicon wafer pieces or gold substrates (as described in the Experimental) were obtained from the precursor polymer 5, because the thus obtained coatings were smoother. Since many technical surfaces do not tolerate THF or strong acids, we coated these surfaces directly with the PZI 1. So far, we established methanol-based coating processes on PDMS (flat surfaces and tubing), polyamide 6.12 (flat surfaces and fibers), polyurethane, silicon, glass, quartz, titanium oxide and iridium oxide. In each case, these technical surfaces were pre-treated by plasma cleaning to generate OH groups on the surface, and then reacted with a benzophenone silane (as described in the Experimental). For example, we could directly coat the entire porous surface of a polyurethane-based wound dressing foam with the PZI 1 (Figure 2 – for this purpose, the PZI was dye-labeled with a covalently bound blue-fluorescent coumarin dye.)

Scheme 2. Surface-attached polymer networks were obtained by spin-coating a solution containing PZI 1 (or its protected analogue 5) and the tetra-functional thiol cross-linker 7 onto benzophenonefunctionalized substrates (e.g. 8), followed by UV irradiation. Simultaneous UV-activated CH-insertions between the polymer and benzophenone, and thiol-ene-reactions between the polymer and the tetrathiol yielded the surface-attached polymer network 6 (or its protected version).

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HS

HS

O n

O O

–

+

O

O

O O HS

1

O O

+ NH 3

O

O O

7

O

SH

O

UV

O

O Si O O

8 S

S

O

O

O

O

O O O

O

O SH

SH O

6 O

a.

b.

c.

d.

Si O O

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Figure 2. Commercially available polyurethane (PU) wound dressing foam coated with PZI: a. photograph of a piece of PU foam (thickness 5 mm); b. optical micrograph of the uncoated PU foam showing the porous structure; c. fluorescence micrograph of the uncoated PU foam (exposure time 100 ms); d. fluorescence micrograph of the PZI-coated PU foam (exposure time 10 ms; for this image, the Coumarinlabeled PZI used).

To evaluate the physical and biological properties of the PZI coating, it was compared it to two structurally similar reference materials: a polyzwitterionic poly(oxonorbornene sulfobetain) (PSB, Figure 1), which has previously shown excellent protein-repellency47 but is not antimicrobial, and a polycationic SMAMP (Figure 1),24 which is intrinsically antimicrobial but protein-adhesive.39 Unlike the PZI, PSB consists of two permanent charges and is thus less affected by pH changes than the COO- and NH3+ groups of the PZI. SMAMP, on the other hand, also carries a NH3+ group but no carboxylate. These reference polymers were immobilized as surface-attached networks as described above for the PZI (see Experimental). Physical characterization. All three surface-attached polymer networks – PZI, PSB and SMAMP – were thoroughly characterized. The thickness the networks (measured by ellipsometry) ranged from 71 to 152 nm (Table 1). FTIR spectra of the networks (SI, Table S1) confirmed that the expected IR bands were present. In particular, both PZI and SMAMP had strong signals corresponding to C=O stretching vibrations of the ester bonds at around 1750 cm-1. In the Boc-protected version of PZI and SMAMP, the signal caused by the presence of the N-H stretching vibration at 3400 cm-1 (from the NH-Boc-group) vanished after deprotection. Due to their structural similarity, the FTIR spectra of the protected PZI and SMAMP differed only slightly in the finger print region. The additional alkyl group in the SMAMP gave rise to more peaks for the SMAMP around 3000 cm-1, and the region above 3000 cm-1 gave slightly different signals due to differences in hydrogen bonding. The most distinct functional group of the PSB was the carbonyl signal from the imide group (1710 cm-1), and the strong absorption around 1200 cm-1 from the the S=O stretching vibration. FTIR spectroscopy was also used to test the stability of PZI in aqueous media. The spectra of PZI networks directly after deprotection, after 1 day in buffer, and after 3

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days in buffer are shown in Figure S1 of the supporting information. No change in the carbonyl peak region was observed, indicating that the ester bonds of the PZI did not hydrolyze to acid, nor did they react with the primary ammonium groups (unlike the SMAMPs, which were previously found to undergo intramolecular transamidation reactions).52 Thus, the PZI coatings were full stable to hydrolysis in the time frame of our investigations. Table 1. Physical characterization data for the surface-attached PZI, SMAMP and PSB networks. The dry layer thickness was measured by ellipsometry; the swellability ratio and the amount of adhered protein were measured by surface plasmon resonance spectroscopy (SPR); the ζ potential parameters including were obtained by electrokinetic measurements; a) data point extrapolated from the curve. Dry Layer Thickness / nm

Swellability Fibrinogen adhesion / Ratio ng mm-2 / H2O

ζmax

Iso-

ζphys

/ mV

electric point

/ mV

pK

PZI

86 ± 1

1.9

0

48 ± 5

6.6 ± 0.1

- 23 ± 5

6.8 ± 0.1

SMAMP

152 ± 1

1.2

8±2

86 ± 5

7.3 ± 0.1

-2±5

7.2 ± 0.1

-34 ± 5

3.1 ± 0.1

PSB

71 ± 3

1.6

0

n/a

2.4

a)

Contact angle measurements (SI, Table S2) indicated that the PZI surface was highly hydrophilic – more hydrophilic than either PSB or SMAMP. The SMAMP network was more hydrophobic than both polyzwitterions due to its butyl group, and because it has only one charge per repeat unit. PZI is more hydrophilic than PSB because the latter has an alkylated cation and a butyl spacer between the cation and the sulfonate. Atomic force microscopy height and phase images (SI, Table S3) used to describe the morphology of the three networks showed that the substrates were homogeneous covered with the networks. PZI had a granular morphology, with an average roughness of 5 nm; the SMAMP surface was the smoothest (roughness 2 nm, with a negligible amount of nano-sized cracks). The PSB surface also had a granular morphology and was quite rough (roughness 19 nm), probably as a result of strong interandintramolecular aggregation of the ion pairs.

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Photoelectron spectroscopy (XPS) measurements of the PZI, SMAMP and PSB coatings immobilized on silica wafers confirmed the presence of the respective polymer networks. The XPS overview spectrum is shown in Figure S3 in the supporting information. For PZI and SMAMP, silicon could not be detected by XPS, indicating full surface coverage. For PSB, a negligible amount of Si could be detected, which is in line with the rougher surface morphology (roughness 19 nm) of PSB and the overall lower coating thickness (71 nm) of that material, which is apparently giving access to a small area fraction of bare Si surface. The parts of the spectra contain the carbon, nitrogen, and oxygen 1s photo electrons are shown in Figure 3. The elemental composition of the SMAMP, PZI and PSB surfaces were calculated from the XPS spectra and compared to the expected elemental composition calculated from the molecular structure of the three polymers (Table 2). As that data indicates, the experimentally determined carbon content of SMAMP was bigger than that of PZI and PSB, respectively. The same trend is found in the calculated carbon content, however the numerical values differ significantly (Ccalculated < CXPS for all polymers). This cannot be attributed to the presence of the tetrathiol cross-linker 7 in the network, because that compound has a calculated carbon content of only 43.2%. However, it is possible that the hydrophobic parts of these amphiphilic polymers segregate to the surface, so that the carbon content of the top few nanometers of each surface, which are probed by XPS, is higher than expected. Nevertheless, the XPS data confirms the presence of the respective polymers on the surface and thus complements the FTIR, ellipsometry, contact angle and AFM results.

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SMAMP PZI PSB

3000 2000 1000 0 3000

c/s

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2000 1000 0 3000

O1s

2000

C1s N1s

1000 0 540

535

415 410 405 400 295

290

285

280

Binding Energy / eV Figure 3: Photoelectron (XPS) spectra (counts per second vs binding energy) of SMAMP, PZI and

PSB. The peaks are labeled with the respective electronic transitions in the PSB spectrum; the other spectra can be assigned analogously.

The surface zeta potential of the PZI, SMAMP and PSB networks was determined by measuring the streaming potential and the pH-dependency of the streaming potential53 for each material (Figure 4). We fitted these titration curves as described in reference

24

using a standard fit for sigmoidal curves.3 From

that fit, we obtained the characteristic points of the curve, i.e. the maximum zeta potential at low pH,

ζ , and the isoelectric point (IEP). The acid constant was obtained from the equation =

ζ + 0.4343

ζ !

, where ζ is the pH value at the half maximum value of ζ , and

the second term (containing Faraday’s constant C, the universal gas constant D and the absolute temperature E) corrects for the ionic strength.48 The zeta potential under physiological conditions (ζFG> at = 7.4) was also estimated. The fitting parameters are summarized in Table S4 in the supporting 3

ζ = ζ + (ζ − ζ ) . This equation has four fitting parameters: ζ , the extrapolated start value of the fitted positive plateau; ζ , the extrapolated end value of the fitted negative plateau; H, the point of inflection and I, a measure of the width and steepness of the sigmoidal curve.

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information, the data obtained is included in Table 1. As expected, ζ was lower for the polyzwitterionic PZI and PSB than for the polycationic SMAMP. ζFG> was approximately zero for the SMAMP, and distinctly negative for PZI and PSB. Importantly, the value of the PZI was also slightly lower than that of the structurally similar SMAMP, which matches previous reports on SMAMPs where a higher acidity also correlated with a higher antimicrobial activity.24 The swellability (= swollen layer thickness/dry layer thickness) of the three polymer networks was determined by surface plasmon resonance spectroscopy (SPR, see Table S5 in the SI). As should be expected due to its higher hydrophilicity, the swellability of the PZI was significantly higher than that of the SMAMP, and also higher than that of the PSB network. This is consistent with the contact angle (CA) data (Table S2), where CA (PZI) < CA (PSB) < CA (SMAMP). Besides determining the swellability of each material, SPR was also used to investigate the protein-repellency of the three polymer networks. In this experiment, each polymer network was exposed to a solution containing fibrinogen under physiological conditions. The kinetics of protein adsorption on the networks was monitored on-line by recording changes of reflectivity as a function of time (SI, Table S6). In addition to this relative measurement, the mass of the adsorbed protein per unit area was also determined quantitatively by measuring the reflectivity curve of the dry networks before and after protein adhesion. Fits to these curves gave the dry thickness of each network before and after the protein adhesion experiment, from which the average layer thickness of adhered protein, and thus the average adsorbed mass per unit area, could be calculated (SI, Table S6). The amount of adsorbed fibrinogen thus obtained is also included in Table 1. The data shows that both PZI and PSB were strongly protein-repellent, with protein adhesion below the detection level of the method (which is < 0.1 ng mm-2), while 8 ng mm-2 of fibrinogen was adsorbed on the SMAMP network.

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Figure 4. Zeta potential titration curves (ζ versus ) for PZI, PSB, and SMAMP, together with the respective data fits.

Table 2. XPS data for the surface-attached PZI, SMAMP and PSB networks, compared to the elemental composition of these polymers calculated from their respective molecular structures.

Elemental compositions (XPS) C 1s

N 1s

O 1s

PZI

67.06

5.04

SMAMP

72.23

PSB

62.05

S 2p

Elemental composition (calc.) C

N

O

S

27.9

52.9

6.2

35.2

0

3.35

24.43

59.1

4.9

28.1

0

2.46

25.96

51.6

7.5

25.8

8.6

9.54

Biological Characterization. To assess the antimicrobial activity of the PZI, SMAMP and PSB networks, two microbiological assays were used: an antimicrobial activity assay on surfaces, which quantifies the amount of bacteria killed by a surface (compared to a reference) after defined contact times (time-killassay),24, 49, 50, 54 and a biofilm formation assay, which determines the amount of bacterial growth on a surface under static conditions in the presence of nutrients over time (up to 72 h). Time-kill experiments

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were performed using standardized procedures, as described in the experimental.

24, 49, 50, 54

In short, the

test surfaces were sprayed with either Gram negative E. coli bacteria, or Gram positive S. aureus bacteria (at 106 bacteria per cm3). After incubating the bacteria on the surfaces for different times, the surviving bacteria were plated out on agar plates, where they were further incubated and then counted. This gave the number of colony forming units (CFUs) over time (Figure 5, the percentage of CFUs relative to a growth control is plotted versus incubation time). This data can be used to compare the relative activity of each surface. The data in Figure 5 indicates that both PZI and SMAMP were highly active against S. aureus (Figure 5a). Surprisingly, PZI killed S. aureus even faster and more quantitatively than the strongly antimicrobial24 SMAMP. While there was a residual growth of 3.7 % on the SMAMP after 4 h, the growth on PZI exposure was 0.0 % (i.e. at or below the detection limit of the method). Both SMAMP and PZI quantitatively killed E. coli bacteria (Figure 5b, 0.0 % growth after 4 h). The protein-repellent PSB, on the other hand, had growth rates > 100 % for both bacteria for all time points studied and was thus, as expected, not antimicrobial.

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Figure 5: Antimicrobial activity assay: time-dependent killing of S. aureus (a.) and E. coli (b.) by PZI, SMAMP and PSB (% bacterial growth, expressed as percentage of colony forming units vs. time). Test samples were normalized to an uncoated silicon wafer as growth control (= 100 % growth). A silicon wafer treated with chlorhexidine digluconate was used as positive control (= 0 % growth). PZI (black squares) was strongly antimicrobial and killed E. coli and S. aureus slightly faster than the antimicrobial SMAMP (dark grey diamonds). PSB, the protein-repellent control surface, did not prevent bacterial growth (light grey triangles, only 2 h and 4 h time points were determined).

For the biofilm formation experiments, E. coli and S. aureus bacteria were grown on the polymer networks over 72 hours under static conditions in the presence of nutrients. At defined time points (12, 24, 48 and 72 h), the growing biofilm was stained using the BacLight Bacterial Viability Kit. Using this stain, all bacteria on the surface were labeled with a green-fluorescent dye (SYTO 9), while only the bacteria with damaged membranes were additionally stained by the red-fluorescent propidium iodide (“Dead”

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stain). Representative images of the biofilm growth on PZI, PSB and SMAMP surfaces obtained by this method are shown in Figure S3 and S4 of the SI. To estimate the total biomass formed on each surface quantitatively, the green fluorescence intensity at different time points was measured and normalized by the fluorescence intensity obtained from the biofilm on an untreated silicon control surface (growth control, defined as 100 % growth, Figure 6). For S. aureus (Figure 6a), the data shows that substantially less biofilm formed on the two polyzwitterionic surfaces PZI and PSB, compared to the growth control (NC). PSB, which is not antimicrobial, had 70 % growth after 12 h compared to untreated surfaces, while the antimicrobial PZI had below 3 % biofilm formation after 12 h. The biofilm amount on the proteinadhesive SMAMP was > 300 % after 12 h. The relative amount of biofilm on PSB decreased with time, indicating that the mass of biofilm on that surface remained low, while the biofilm mass on the growth control kept increasing. On the antimicrobial PZI, the initially very low relative bacterial growth increased to about 30 % after 48 h, and then decreases, too. The biofilm growth of E. coli (Figure 6b) was significantly reduced on all polymer networks compared to the negative control. SMAMP and the PZI had similarly low biofilm growth rates when exposed to E. coli, while the non-antimicrobial PSB was slightly more covered with bacteria. Since the biofilm formation data was obtained from the green-fluorescent bacteria, it describes the total amount of bacteria on each surface, but not whether or not they are affected by the underlying surface. The latter can be estimated qualitatively from the red-green overlay of fluorescence microscopy images obtained from the test surfaces (SI, Figures S3 and S4). These revealed, especially in the case of S. aureus (SI, Figure S3), that a significant amount of the bacteria on the polycationic SMAMP surface were dead (red), which is consistent with the antimicrobial activity data. On the other hand, most of the bacteria that adhered on the PSB were intact (green). The small amount of bacteria that adhered to PZI was also predominantly red after 48 h and 72 h, indicating that PZI did not only reduce the number of adhering bacteria, but also killed the ones that managed to adhere. Thus, the biofilm formation data is consistent with the antimicrobial activity data. It is also consistent with the protein-adhesion data: SMAMP, which

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was protein-adhesive, was also strongly adhesive for bacteria and the debris of dead bacteria. On the other hand, bacteria could only weakly adhere to the protein-repellent PSB and PZI.

Figure 6: Relative amount of biofilm formation (percentage of bacterial growth relative to a blank wafer (defined as 100 % growth) by S. aureus (a.) and E. coli (b.) on PSB, PZI and SMAMP networks after 12, 24, 48 and 72 h.

To judge the toxicity of the PZI towards human cells, three assays were used. The metabolic activity of immortalized human keratinocytes grown on the PZI, SMAMP and PSB surfaces was quantified using the Alamar Blue assay,55 a standard cell toxicity assay. Optical microscopy (phase contrast) was used to assess the morphology and density of the keratinocytes grown on PZI and PSB, and a Live-Dead assay for mammalian cells (green-fluorescent SYTO16/red-fluorescent propidium iodide) was used to qualitatively

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visualize the relative amount of membrane-compromised (red, “dead”) and healthy (green) keratinocytes. Additionally, the PZI concentration that causes lysis of 50% of human red blood cells (HC50), a method to assess toxicity in solution, was determined. For the Alamar Blue assay,55 immortalized, non-carcinogenic human keratinocytes were seeded on the test surfaces and cultivated for 24 h, 48 h, and 72 h, respectively. Since healthy cells metabolize the Alamar Blue dye, relative dye reduction (normalized to various controls) is an indication of cell viability. The relative dye reduction data obtained for keratinocytes grown on PZI, SMAMP and PSB is shown in Figure 7. (Absolute values can be found in Figure S6 in the SI.)

Figure 7: Relative reduction of Alamar Blue dye (normalized to the growth control (= 100 %)) by human keratinocytes grown on PZI, SMAMP and PSB for 24, 48 and 72 h, respectively.

The dye reduction data indicates that the keratinocytes grew about equally well on PZI, PSB and regular cell culture substrates, given the experimental error of the method. The cell growth on SMAMP, on the other hand, was significantly diminished, which indicates a reduced metabolic activity of these keratinocytes.

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Optical micrographs (SI, Figure S5) indicated that there were no significant differences in the cell morphology or the cell density of human keratinocytes grown on PZI, PSB, and the growth control. Fluorescence microscopy images confirmed this (Figure 8), and moreover indicated that most of these cells were viable (green), with approximately the same low amount of red (= membrane-compromised) keratinocytes on PZI, PSB and the growth control (Figure 8). For the SMAMP, on the other hand, significant membrane-damage of mammalian cells in contact with the SMAMP was observed,24,

28

including an up-regulation of the IL6 RNA marker for inflammation in keratinocytes grown on SMAMP surfaces, indicating partial toxicity.24 The HC50 value determined for the PZI was 8000 ± 500 µg mL-1, which compares to a HC50 value of 10 µg µL-1 for the SMAMP.45 Thus, all toxicity data indicate that the PZI is highly cell compatible in solution and on surfaces, similarly to the PSB reference surface. SMAMP, with comparable antimicrobial activity, was partially toxic.

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Figure 8. Immortalized human keratinocytes grown on PZI and PSB after 72 h. Uncoated glass slides were used as growth control (- control); isopropanol had been added to the keratinocytes on the + control after 72 h. The scale bar ( = 100 µm) is identical for all images. The green stain (SYTO 16, first column) visualizes the overall cell density of the keratinocytes on each surface, the red stain (propidium iodide, second column) indicates the relative number of membrane-compromised cells. The third colunm is an overlay of the green and red images.

Discussion. Polyzwitterions are highly interesting molecules whose protein-repellency and biofilm-resistance on surfaces has been intensively studied,16, 18, 47, 56-59 but so far, no permanently antimicrobially active, non-

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leaching polyzwitterion has been reported. However, substantial efforts have been put in the synthesis of simultaneously active antimicrobial and protein-repellent materials – for example, polyzwitterionic coatings leaching antimicrobial components,60 polymer surfaces with antimicrobial and protein-repellent components, or surfaces that are switchable from an antimicrobial to a zwitterionic state43 have been synthesized. In comparison to many of these materials, the synthesis and surface-immobilization of the here presented PZI is rather straight-forward. The obtained surface-attached PZI networks near-quantitatively killed S. aureus and E. coli (as representatives of Gram-positive and Gram-negative bacteria, respectively), had a protein adsorption level that was below the sensing threshold of surface plasmon resonance spectroscopy, and reduced the biofilm formation of S. aureus and E. coli in long-term experiments under static conditions more drastically than the non-fouling PSB control material. Compared to the cationic antimicrobial SMAMP polymer, the biofilm reduction of S. aureus by PZI was most evident. The cationic SMAMP polymer had S. aureus growth rates of > 300 % after 12 h, while that of the PZI was < 3 % (normalized to the control surface). This is an indication that the debris of the dead bacteria cannot adhere as firmly to the PZI surface as it can stick to the polycationic SMAMP surface. These results match the protein adhesion data for PZI, SMAMP, and PSB respectively, and thus confirm the well-known fact that protein-adhesion on a surface is a prerequisite for biofilm formation.61 The Live-Dead stain of bacteria grown on PZI, SMAMP and PSB indicated that the few bacteria adhered on PZI were predominantly red (= membrane compromised), while those on PSB were mostly green (= membrane intact). This is a significant difference and indicates that bacteria adhered to PZI cannot proliferate, while those attached on PSB are viable and can initiate biofilm formation. This explains the higher biofilm formation rates on PSB compared to PZI. It also confirms the hypothesis that materials with two lines of defense against bacteria39 would be holding up longer against biofilm formation than only non-fouling or only antimicrobial materials.

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When comparing the physical properties of PZI, SMAMP, and PSB, the most striking difference between the three polymer surfaces are the pronounced differences in their zeta potential. Under pseudophysiological conditions, PZI and PSB had a negative zeta potential, while the SMAMP was nearly charge-neutral. This is somewhat unexpected when considering the chemical structures of the polymers, but can be explained by the fact that counterions adsorb on all three surfaces. Since anionic counterions tend to adsorb more firmly than cations,53 the zeta potential of each material becomes more negative than expected from the nominal charge. Thus, while negatively charged bacteria and cell debris can still adhere to the positive charge on the SMAMP under physiological conditions, they cannot adhere as easily to the PZI and PSB, so that a significantly lower amount of protein adhesion and biofilm formation is observed on these surfaces, compared to SMAMP. Yet the surface charge does not seem to be the only critical factor for antimicrobial activity(as was observed previously),24 since PSB does not kill bacteria, whereas PZI does. Another factor could be the nature and the pH-responsiveness of the functional groups present. Both PZI and SMAMP consist of functional groups which make them pH-responsive, while PSB consists of two permanent charges and is thus always zwitterionic. Also, the values of PZI (6.8) and the antimicrobial SMAMP (7.2) differ only by 0.4 pH units. PZI is in fact similar to the amino acid alanine, which has a value of 6.1 and is mostly zwitterionic under physiological conditions. Certain poly(alanines) have been recently reported to be antibacterial in solution.62 Thus, the antimicrobial activity of the PZI may be explained by its low value, which is comparable to that of the SMAMP. Since it is well-known from the field of contact-active antimicrobial polymers that these polymers need to be cationic to be active, our current working hypothesis for the PZI activity is that there are local fluctuations in the surface the charge distribution of the PZI occurring when the bacteria adhere to the PZI. In other words, areas with temporary excess of NH3+ over COO- may exist, making the PZI surface temporarily sufficiently charged locally to kill adhering bacteria. At present, this is purely speculative because no method known to us can be used to prove such a local fluctuation. Indeed, it is difficult to directly visualize the antimicrobial effect of the surface-attached polymers on adhering bacteria. In the case of

39


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solution-born antimicrobial peptides or SMAMPs, the entire cell envelope of the bacterial cell is covered with the active agent, so their effects on the cell membrane can be either visualized by AFM62 or electron microscopy.45 For contact active surfaces, however, the action of the antimicrobial agent is limited to the surface-bacteria contact area, which is not accessible to an AFM tip.63 Thus, surface-bacteria interactions have to be assessed indirectly, for example through the above described antimicrobial activity assay and the life-dead-stain used in the biofilm experiments. Even if the proposed charge fluctuation could be demonstrated experimentally, the remaining question would be why such a postulated charge fluctuation only affects bacteria (i.e. kills them), while the PZI remains protein-repellent, repellent for cell-debris, and promotes adhesion and growth of keratinocytes. Again, we have to speculate on this. One could assume that the underlying cause is a change in pH caused by the viable adhered bacteria. It is well-known that many bacteria secrete acids as their metabolites. If this was the case for surface-attached bacteria on PZI, it would locally change the pH, and thus the local charge of the PZI underneath these, making them cationic. However, the effect would directly stop as soon as the bacterial metabolism is stopped (due to the neutralizing effect of the surrounding medium). Thus, the PZI surface would become neutral again once the bacteria are killed, and the bacterial debris would not adhere. Neither would proteins adhere, as it does not affect the local pH value. On the other hand, while the presence of positive charges may assist the adhesion of mammalian cells like keratinocytes to surfaces, this is not prerequisite (the membranes of mammalian cells themselves consist of zwitterionic phospholipids). Their proliferation mainly depends on the presence of calcium, epidermal growth factor and hydrocortisone. Thus, the surfaces remain compatible with human cells like keratinocytes despite their antimicrobial activity and cell compatibility.

Conclusion. A polyzwitterion (PZI) that is simultaneously antimicrobial, protein-repellent and cell-compatible, and reduces biofilm formation is presented. It was synthesized in a few steps and could be easily surface-

40


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attached to laboratory and real live surfaces. Importantly, both the PZI and the N-boc protected PZI precursor were soluble in organic solvents. Thus, surface-attached PZI networks could be obtained by common surface coating techniques like spin-coating and dip-coating. This is of particular relevance for practical applications and was demonstrated by coating a commercially available wound dressing with a fluorescent version of the PZI. By comparison of the PZI with a structurally similar, protein-repellent but non-antimicrobial poly(sulfobetaine) (PSB), and an antimicrobial but protein-adhesive polycationic SMAMP, it was demonstrated that the PZI indeed united the benefits of both reference materials: The surface-attached PZI near-quantitatively killed S. aureus and E. coli sprayed on the coated surface - in the case of S. aureus, it did so even more efficiently that the highly antimicrobial SMAMP. Yet the protein-adhesion level of PZI was below the detection level of surface plasmon resonance spectroscopy, as for the protein-repellent reference PSB. PZI also reduced the biofilm formation by S. aureus and E. coli in the presence of nutrients as efficiently as the PSB. Additionally, it was fully compatible with human keratinocytes in various cell assays. These properties – antimicrobial activity, protein-repellency, prevention of biofilm formation and cell compatibility – in a single material are a hard-to-achieve yet highly desired combination for the medical field. Thus, the here reported material could be of importance for applications like wound dressings and catheter coatings. Of course, properties relevant for practical applications, such as shelf-life, stability under application conditions, and sterilizability still have to be determined. If these are also favorable, the material may be a true asset in the fight against biofilms on medical devices.

Acknowledgement Funding for this project from the Deutsche Forschungsgemeinschaft (Emmy-Noether-Program, LI1714/5-1) and the Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg (GenMik II) is gratefully acknowledged. 41


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Dorner, F.; Lienkamp, K., CHAPTER 5 Polymer-Based Synthetic Mimics of Antimicrobial Peptides (SMAMPs) - A New Class of Nature-Inspired Antimicrobial Agents with Low Bacterial Resistance Formation Potential. In Polymeric Materials with Antimicrobial Activity: From Synthesis to Applications, The Royal Society of Chemistry: 2014; pp 97-138. Lienkamp, K.; Madkour, A. E.; Musante, A.; Nelson, C. F.; Nusslein, K.; Tew, G. N., J. Am. Chem. Soc. 2008, 130, (30), 9836-9843. Lienkamp, K.; Tew, G. N., Chem. Eur. J. 2009, 15, (44), 11784-11800. Brogden, K. A., Nature Rev. Microbiol. 2005, 3, (3), 238-250. Brogden, N. K.; Mehalick, L.; Fischer, C. L.; Wertz, P. W.; Brogden, K. A., Skin Pharmacol. Physiol. 2012, 25, (4), 167-181. Zasloff, M., Nature 2002, 415, (6870), 389-395. Lienkamp, K.; Kumar, K.-N.; Som, A.; Nuesslein, K.; Tew, G. N., Chem. Eur. J. 2009, 15, 11710-11714. Lienkamp, K.; Madkour, A. E.; Kumar, K.-N.; Nuesslein, K.; Tew, G. N., Chem. Eur. J. 2009, 15, (43), 11715-11722. Rodrigues, L. R., Adv. Exp. Med. Biol. 2011, 715, 351-367. Jewrajka, S. K.; Haldar, S., Polym. Compos. 2011, 32, (11), 1851-1861. Hartleb, W.; Saar, J. S.; Zou, P.; Lienkamp, K., Macromol. Chem. Phys. 2016, 217, (2), 225-231. Zou, P.; Hartleb, W.; Lienkamp, K., J. Mater. Chem. 2012, 22, (37), 19579-19589. Wu, H.-X.; Tan, L.; Tang, Z.-W.; Yang, M.-Y.; Xiao, J.-Y.; Liu, C.-J.; Zhuo, R.-X., ACS Appl. Mater. Inter. 2015, 7, (12), 7008-7015. Tang, Z.-W.; Ma, C.-Y.; Wu, H.-X.; Tan, L.; Xiao, J.-Y.; Zhuo, R.-X.; Liu, C.-J., Progr. Organ. Coat. 2016, 97, 277-287. Mi, L.; Jiang, S., Angew. Chem., Int. Ed. 2014, 53, (7), 1746-1754. Gianneli, M.; Roskamp, R. F.; Jonas, U.; Loppinet, B.; Fytas, G.; Knoll, W., Soft Matter 2008, 4, , 1443-1447. Al-Ahmad, A.; Laird, D.; Zou, P.; Tomakidi, P.; Steinberg, T.; Lienkamp, K., PLoS One 2013, 8, (9), e73812. Lienkamp, K.; Madkour, A. E.; Musante, A.; Nelson, C. F.; Nüsslein, K.; Tew, G. N., J. Am. Chem. Soc. 2008, 130, (30), 9836-9843. Colak, S.; Tew, G. N., Langmuir 2012, 28, 666-675. Jacobasch, H.-J., Progr. Organ. Coat. 1989, 17, (2), 115-133. Al-Ahmad, A.; Zou, P.; Guevara-Solarte, D. L.; Hellwig, E.; Steinberg, T.; Lienkamp, K., PLoS One 2014, e111357. Haldar, J.; Weight, A. K.; Klibanov, A. M., Nat. Protoc. 2007, 2, (10), 2412-2417. Rennie, J.; Arnt, L.; Tang, H. Z.; Nusslein, K.; Tew, G. N., J. Ind. Microbiol. Biotechnol. 2005, 32, (7), 296-300. Dorner, F.; Boschert, D.; Schneider, A.; Hartleb, W.; Al-Ahmad, A.; Lienkamp, K., ACS Macro Lett. 2015, 4, 1337-1340. Luxbacher, T., The Zeta Guide - Principles of the streaming potential technique. 1st ed.; AntonPaar GmbH: Graz, 2014; p 135. Gabriel, G. J.; Madkour, A. E.; Dabkowski, J. M.; Nelson, C. F.; Nüsslein, K.; Tew, G. N., Biomacromolecules 2008, 9, (11), 2980-2983. 43


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O

O –O

O

H3 N

+

O

n

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Paragon Plus Environ 16 17

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

Figure 1: Structure of the simultaneously antimicrobial and protein-repellent polyzwitterion (PZI) and two reference molecules, the antimicrobial, protein-adhesive SMAMP and the protein-repellent, non-antimicrobial PSB. 254x190mm (96 x 96 DPI)


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4 DMAP

3

2

HCl

1

5


G3

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1

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7

UV

6 ACS Paragon Plus Environment

8

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a.

b.

c.

d.

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285

Binding Energy / eV

415 410 405 400 295 535 0 540

1000

2000

0 3000

1000

2000

0 3000

1000

2000

3000

O1s

N1s

290

C1s SMAMP PZI PSB

c/s

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Figure 5: Antimicrobial activity assay: time-dependent killing of S. aureus (a.) and E. coli (b.) by PZI, SMAMP and PSB (% bacterial growth, expressed as percentage of colony forming units vs. time). Test samples were normalized to an uncoated silicon wafer as growth control (= 100 % growth). A silicon wafer treated with chlorhexidine digluconate was used as positive control (= 0 % growth). PZI (black squares) was strongly antimicrobial and killed E. coli and S. aureus slightly faster than the antimicrobial SMAMP (dark grey diamonds). PSB, the protein-repellent control surface, did not prevent bacterial growth (light grey triangles, only 2 h and 4 h time points were determined).

254x190mm (96 x 96 DPI)


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Figure 6: Relative amount of biofilm formation (percentage of bacterial growth relative to a blank wafer (defined as 100 % growth) by S. aureus (a.) and E. coli (b.) on PSB, PZI and SMAMP networks after 12, 24, 48 and 72 h. 254x190mm (96 x 96 DPI)


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% Dye Reduction

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140 120 100 80 60 40 20 0

24 h 48 h 72 h

PZI

SMAMP

PSB


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Figure 8. Immortalized human keratinocytes grown on PZI and PSB after 72 h. Uncoated glass slides were used as growth control (- control); isopropanol had been added to the keratinocytes on the + control after 72 h. The scale bar ( = 100 µm) is identical for all images. The green stain (SYTO 16, first column) visualizes the overall cell density of the keratinocytes on each surface, the red stain (propidium iodide, second column) indicates the relative number of membrane-compromised cells. The third colunm is an overlay of the green and red images.

128x133mm (300 x 300 DPI)


A Simultaneously Antimicrobial, Protein-Repellent ... - ACS Publications

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