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Antifouling and antibacterial multi-functional polyzwitterion / enzyme coating on silicone catheter material prepared by electrostatic layer-by-layer assembly Anne Vaterrodt, Barbara Thallinger, Kevin Daumann, Dereck Koch, Georg M. Guebitz, and Mathias Ulbricht Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04303 • Publication Date (Web): 14 Jan 2016 Downloaded from http://pubs.acs.org on January 25, 2016

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Antifouling and antibacterial multi-functional polyzwitterion / enzyme coating on silicone catheter material prepared by electrostatic layer-by-layer assembly

Anne Vaterrodta, Barbara Thallingerb, Kevin Daumanna, Dereck Kocha, Georg M. Guebitzb,c, Mathias Ulbrichta,*

a

Lehrstuhl für Technische Chemie II, Universität Duisburg-Essen, 45117 Essen, Germany

b

Institute of Environmental Biotechnology, BOKU - University of Natural Resources and Life

Sciences, 3430 Tulln, Austria c

Austrian Centre of Industrial Biotechnology ACIB, Konrad Lorenz Strasse 20, 3430 Tulln,

Austria * corresponding author: [email protected]

Keywords: antibiofilm, antimicrobial, cellobiose dehydrogenase, zwitterionic polymer, urinary catheter, polydimethylsiloxane, layer-by-layer assembly

Abstract The formation of bacterial biofilms on indwelling medical devices generally causes high risks for adverse complications such as catheter associated urinary tract infections. In this work, a strategy for innovative coatings of polydimethylsiloxane (PDMS) catheter material, using layer-by-layer assembly with three novel functional polymeric building blocks is reported, i.e., an antifouling copolymer with zwitterionic and quaternary ammonium side groups, a contact biocidal derivative of that polymer with octyl groups and the antibacterial hydrogen peroxide (H2O2) - producing enzyme cellobiose dehydrogenase (CDH). CDH oxidizes oligosaccharides transferring electrons to oxygen resulting in the production of H2O2. The design and synthesis of random copolymers which combine segments that have antifouling properties by zwitterionic groups and can be used for electrostatically driven layer-by-layer (LbL) assembly at the same time were based on atom transfer radical polymerization of dimethylaminoethyl methacrylate and subsequent partial sulfobetainization with 1,3propanesultone followed by quaternization with methyl iodide only or octyl bromide and thereafter methyl iodide. The alternating multilayer systems were formed by consecutive adsorption of the novel polycations with up to 50% zwitterionic groups and of poly(styrene 1 ACS Paragon Plus Environment

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sulfonate) as polyanion. Due to its negative charge the enzyme CDH was also firmly embedded as polyanionic layer in the multilayer system. This LbL coating procedure was first performed on pre-functionalized silicon wafers and studied in detail with ellipsometry as well as contact angle (CA) and zetapotential (ZP) measurements before it was transferred to pre-functionalized PDMS and analyzed by CA and ZP measurements as well as atomic force microscopy. The coatings comprising six layers were stable and yielded a more neutral and hydrophilic surface compared to PDMA, the polycation with 50% zwitterionic groups had the largest effect. Enzyme activity was found to be dependent of the depth of embedment in the multi-layer coating. Depending on the used polymeric building block, up to 60% reduction of amount of adhering bacteria and clear evidence for killed bacteria due to the antimicrobial functionality of the coating could be confirmed. Overall, this work demonstrates the feasibility of an easy to perform and shape-independent method to prepare an anti-fouling and anti-microbial coating for significant reduction of biofilm formation and thus reducing the risk of acquiring infections by using urinary catheters.

1. Introduction On any surface which is exposed to water, organic compounds and microorganisms attach in a short period of time. The result after a few hours is the formation of a complex structure which is called a biofilm. Fouling on surfaces is a known problem in numerous biomedical applications such as prosthetic devices, implants, contact lenses and catheters. For the latter the adhesion of microorganisms on catheters and the associated risk of an infection is one of the most common complications in hospitals. Currently about 40% of all bacterial infections occurring in hospitals are found to be catheter-associated urinary tract infection, what considerably increases the healthcare costs, the length of stay in the hospital and the antibiotic use

1–3

. To prevent infections it is urgently needed to design surfaces that inhibit

bacterial colonization and thus reduce the occurrence of biofilm formation. The most commonly used approaches are antimicrobial and antifouling coatings 4. For antimicrobial surface functionalization of implants substances such as antibiotics, silver and nitric oxide as active components to be released from the coating are often reviewed 5– 7

. However, the main problems of these coatings are the toxicity and the increasing

resistance to antibiotics 8.

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Contact-biocidal coatings based on surface-immobilized quaternary ammonium compounds (QAC) had become an excellent approach to render the surface antiseptic

9,10

. The long

hydrophobic polycationic chains are able to penetrate microbial cell envelopes and kill the microbes. Klibanov et al. have reviewed the most important aspects of these strategies 11,12. The advantage is that no QAC is released, the downside of this method is the attachment of the dead microbes on the QAC surface which is problematic because they change the surface properties and trigger the adhesion of more microorganisms 13. Recently, new antimicrobial systems, i.e. enzyme-based antifouling coatings, had been introduced. Enzymes like glucose oxidase and cellobiose dehydrogenase (CDH) lead to the generation of hydrogen peroxide [H2O2] which is well-known as antimicrobial agent 14. They also generally exhibit good biocompatibility, broad spectrum antimicrobial activity and are less likely to evoke the risk of bacterial resistance and inadequate efficiency 6,15. Another strategy to prevent non-specific adsorption and adhesion of cells and microorganisms is to create a low fouling surface due to anti-adhesive properties. As such antifouling materials poly(ethylene glycol) (PEG) derivatives or zwitterionic polymers have been extensively used 16–18. It has been found that these polymers form a tightly bound and structured water layer which creates a thermodynamically unfavorable surface for adsorption of organic substances or adhesion of particles 5. In this research, polydimethylsiloxane (PDMS) was chosen as catheter material to establish an antifouling functionalization. PDMS is the current material of choice for urinary catheters due to its advantageous material properties such as non-toxicity, biocompatibility, easy handling and low cost 19. There are two types of PDMS used in the silicone catheter, the tube material and the balloon material, the latter being more elastic. Both types have the same ingredients; the only differences are in the cross-linking density and the amount of added filler. A typical filler is silica, which is for a better stickiness and enhanced mechanical strength and hardness

20

. In the present studies, the balloon material was mainly used

because biofilm formation mostly occurs in the balloon region inside the bladder 21,22. PDMS was pre-modified by surface hydrolysis and amination using a functional silane. Afterwards the surface was coated via layer-by-layer (LbL) assembly, using novel functional cationic copolymers, either an antifouling copolymer with zwitterionic and quaternary ammonium side groups, or a contact biocidal derivative of that polymer with octyl groups.


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As polyanion poly(styrene sulfonate) (PSS) was used because it is a well established and characterized polyanion for LbL assembly 23,24. In addition, the antibacterial enzyme CDH was immobilized in between two layers of cationic copolymers. To obtain the novel polycationic/zwitterionic materials, poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) was synthesized by atomic transfer radical polymerization (ATRP) to obtain adjustable molecular weight and low polydispersity

25

, subsequent partial sulfobetainization with 1,3-

propanesultone yielding poly(sulfobetaine methacrylate) (PSPE) segments, and finally quaternization with methyl iodide alone or octyl bromide and thereafter methyl iodide. The possibility to integrate zwitterionic groups in the cationic layer via LbL assembly was first investigated with copolymers comprising 0%, 25% and 50% sulfobetaine on silicon wafers as model surfaces. A pre-modification of the substrates was realized by the use of 3(aminopropyl)triethoxysilane (APTS) to introduce amino groups to the surface. After transfer to analogously pre-modified PDMS, further studies about stability, enzymatic activity and bacteria deposition and growth were performed in order to evaluate the antifouling and antibacterial properties of the coated catheter material. An overview on the approaches is shown in Figure 1.


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Figure 1: Schematic representation of layer-by-layer assembly on APTS pre-modified silicon (wafer) or silicone (PDMS) surfaces with different polymeric building blocks; polyanions: poly(styrene sulfonate) (PSS) and cellobiose dehydrogenase (CDH) for antibacterial coating; polycations: novel copolymers (PTMAEMA-co-PSPE) with varied sulfobetaine fraction for antifouling properties and optional addition of quaternary hydrophobic groups for contact biocide functionality.

2. Experimental section 2.1

Materials

2-(Dimethylamino)ethyl

methacrylate

(DMAEMA),

iodomethane

(MeI),

ethyl-α-bromoisobutyrate (EBB), 1-bromooctane, (3-aminopropyl)triethoxysilane (APTS), poly(sodium

4-styrenesulfonate)

(PSS;

Mw

~

70

kg/mol),

1,1,4,7,10,10-

hexamethyltriethylenetetramine (HMTETA), copper(I) bromide (CuBr), 1,3-propanesultone, iodomethane, 1-bromooctane, ninhydrin, cellobiose, glucose, crystal violet, sodium azide, sulfuric acid, hydrogen chloride, hydrogen peroxide, acetone, monosodium phosphate, disodium phosphate, toluene, methanol and tetrahydrofuran (THF), dimethylformamid (DMF) were purchased from Sigma-Aldrich. Recombinant M. thermophilum cellobiose

dehydrogenase (CDH) was produced as previously described 26. Synthetic urine according to DIN EN 1616 was manufactured by SYNTHETIC URINE e.K. (Germany). Silicon wafers were obtained from SilChem (Germany). Polydimethyl/vinylmethyl siloxane (PDMS; designated as VMQ polymer by ASTM D 1418) urinary catheter materials flat sheets were supplied by Degania Silicone (Emek Hayarden, Israel). Staphylococcus aureus ATCC 10145 was obtained from the culture collection of the Institute of Environmental Biotechnology (Graz, Austria).

2.2

Polymer syntheses and characterization

An overview on all synthesis steps is given in Figure 2.


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Figure 2: Synthesis of the novel copolymers; (a) ATRP of monomer DMAEMA; (b) sulfobetainization to obtain zwitterionic side groups; (c) optionally quaternization by octyl groups (c); (d) quaternization by methyl groups; to yield PTMAEMA-co-PSPE (k = 0) with varied composition, PTMAEMA-co-PSPE-co-P(octylDMAEMA) or PTMAEMA (k = 0, n = 0) used as reference.

Preparation of PDMAEMA by ATRP of DMAEMA. CuBr (71.72 mg, 0.5 mmol) was introduced into a Schlenk flask equipped with a rubber septum and three freeze-pump-thaw cycles were performed to get rid of trapped oxygen. After oxygen had been removed by degassing the solution with nitrogen, the monomer DMAEMA (11.79 ml; 70 mmol), the ligand HMTETA (140 µl; 0.5 mmol) and toluene (7 ml) were added via syringe. The mixture was stirred and heated to 50°C. Then polymerization was started by addition of EBB (75.76 µl; 0.5 mmol) as initiator dissolved in toluene (7 ml); this solution had before also been degassed with nitrogen. After 300 minutes, the polymerization was terminated by adding THF and exposing the system to air. The solution was passed through a basic aluminum oxid column to remove the catalyst. The resulting solution was concentrated by rotary evaporation and the polymer was recovered by precipitation in cold n-hexane, followed by drying in vacuum at 40 °C for 1 day.

Sulfobetainization of PDMAEMA to zwitterionic PDMAEMA-co-PSPE. The reaction was carried out in a round flask with dry THF (250 ml) as solvent. A stoichiometric amount of 1,3propanesultone, based on the number of DMAEMA units and the targeted fraction of zwitterionic groups, was added into the polymer solution which was then stirred for 1 day at room temperature (cf. 27). Gelation occurred because of the bad solubility of polyzwitterions 6 ACS Paragon Plus Environment

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in THF. The copolymer was recovered and purified by removing the THF, washing with acetone and drying in vacuum at 40 °C for 1 day.

Quaternization of PDMAEMA-co-PSPE to PTMAEMA-co-PSPE derivatives. Two different quaternization reagents, 1-bromooctane and iodomethane, were used. For imparting antimicrobial properties, the copolymer was firstly quaternized with 1-bromooctane in 0.1 mol/L methanol at 60 °C for 48 hours under nitrogen atmosphere. This was only done for PDMAEMA-co-PSPE with 25% zwitterionic groups, and only so much 1-bromooctane was added that only 25% of the PDMAEMA segments were quaternized. For complete quaternization of all DMAEMA groups, the copolymers were dissolved in methanol and a threefold excess of iodomethane relative to the amount of tertiary amino groups in the polymer was added and the solution was stirred at room temperature over night. After each quaternization, the solution was concentrated by rotary evaporation, and the polymer was precipitated in n-hexane and dried in vacuum oven overnight. The chemical composition was characterized using 1H-NMR and IR-spectroscopy.

Polymer Characterization. The monomer conversion and the chemical composition were determined using 1H-NMR spectroscopy. In addition, IR spectroscopy and elemental analysis were used. The relative molecular weight was characterized by size exclusion chromatography (SEC). Details on instrumentation and procedures can be found in Supporting Information (SI).

2.3

Coating preparation

Amination of silicon wafer surfaces. Silicon wafers (9 x 9 mm²) were cleaned by Piranha solution, i.e. concentrated sulfuric acid and hydrogen peroxide (in a volume ratio of 1:1), for 45 minutes, then rinsed extensively with water and ethanol and finally dried under nitrogen. These wafers were then immersed into a 5 wt% APTS solution in dry toluene and heated under reflux and nitrogen atmosphere for 45 minutes (cf. 28). The wafers were finally washed with toluene and ethanol to remove the APTS derivatives. The quality of the prefunctionalization was assessed with the contact angle measurements; data should be in the rage 53°-59° for a perfect APTS monolayer, according to the literature

29

. The presence of

primary amino groups was also confirmed by the ninhydrin test, performed by heating the


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samples in a 2% (w/v) ninhydrin/ethanol solution at 50°C leading to a characteristic color change.

Amination of PDMS catheter material surfaces. The pre-functionalization was also done using APTS and according to literature 30,31. The PDMS strips (3.2 x 1 cm²/radius of 24 mm for ZP measurements) were firstly washed three times in ethanol for 30 minutes and then immersed into a solution of H2O/H2O2/HCl (in a volume ratio of 5:1:1) for 5 minutes to generate hydroxyl groups on the surface

30

. Immediately after oxidation the samples were

transferred to 5 % (v/v) APTS in ethanol for 24 hours at room temperature, then the samples were finally washed three times for 5 minutes in ethanol 31. The existence of primary amino groups on the PDMS surface was confirmed by dropping a 2 % (w/v) ninhydrin ethanol solution onto the surface. After incubation at 60 °C, this lead to a characteristic color change.

Coating via LbL assembly. The APTS-functionalized substrates with their positively charged surface were immersed in a 10-3 mol/L solution of PSS (MW ~ 70 kg/mol) at pH ~ 6 for 10 minutes, followed by two times washing with distilled water for 5 minutes. The PSS-coated substrate was then immersed into a 10-3 M solution of the oppositely charged polyelectrolyte, the novel copolymers PTMAEMA-co-PSPE, at pH ~ 6 for 10 minutes, followed by washing with distilled water two times for 5 minutes to remove non-adherent polyelectrolyte from the surface. This alternate deposition of cationic or anionic polyelectrolyte was repeated three times until a stable layer system was achieved. The last layer was always PTMAEMA-co-PSPE. In order to obtain a coating with contact biocidal properties, for the last (6th) layer the polycation with 25% zwitterionic and 25% octylquaternized groups was used.

Enzyme immobilization. Due to the fact that the enzyme CDH has an isoelectric point at pH = 4.3, it can be used as polyanion for LbL assembly. CDH was dissolved at a concentration of 1 mg/ml in 0.1 M phosphate-buffered saline (PBS), pH = 7.4. The pre-coated substrate was in the 3rd or 5th LbL step immersed instead of PSS into the CDH solution, so that the CDH was firmly

embedded

in

the

layer

system

between

two

layers

of

the

novel

polycation/zwitterions. The coated samples were then either stored in 0.02 M citrate buffer at pH = 5.5 or freeze-dried. For the latter, the samples were rapidly frozen at -196°C and then dried in a freeze dryer (Alpha 1-4 from Christ).

2.4

Physical and physico-chemical characterization of the coatings 8 ACS Paragon Plus Environment

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Ellipsometry was used to monitor the thicknesses of self-assembled layers on silicon wafers using the instrument MM-16 (HORIBA Jobin Yvon). X-ray photoelectron spectroscopy (XPS) was used for chemical composition analysis of the polymer films deposited on silicon wafers with a Versaprobe IITM from Ulvac-Phi. Contact angle (CA) measurements of the LbL assemblies were done using an optical measurement system (OCA 15 Plus, Dataphysics, Germany). Zeta potentials (ZP) of the LbL assemblies were measured by using a SurPASS electrokinetic analyzer (Anton-Paar, Austria). Atomic force microscopy (AFM) using an Autoprobe CP Research or a Nanoscope IIIa, both from Veeco Instruments was used to investigate the topography of the PDMS catheter materials. More details on procedures can be found in SI.

2.5

Functional characterization of the coatings

Enzymatic activity assay: H2O2 production capacity of the immobilized CDH was assessed by the Amplex Red Assay

32

, with some modifications using cellobiose as substrate (cf.

15

).

Biofilm formation assay: S. aureus (ATCC 10145) was used to evaluate the bacterial adherence to the sample surfaces and the crystal violet assay was adopted to quantify the adhering biomass (cf.

15

). Antimicrobial activity assay: Samples were investigated by

incubation with S. aureus in the presence of cellobiose and visualization of attached bacteria using LIVE/DEAD staining and confocal laser scanning microscopy (CLSM; cf. 15). All detailed procedures can be found in SI.

3. Results and discussion 3.1

Copolymer synthesis

The aim of the polymerization step was to obtain a polymer with sufficient size at low polydispersity. For polyelectrolytes with a molecular mass below 10000 g/mol it had been reported that atypical multilayer characteristics can be obtained during the LbL deposition 33,34

. The total multilayer growth may be inhibited by the weak attachment of

polyelectrolytes. Glinel et al.

35

investigated the influence of the cationic charge density of

polyelectrolytes on the growth of the multilayer system. They worked with the highly charged PSS and different charge density of the cationic polyelectrolyte, obtained by the systematic variation of the diblock copolymer of poly(diallyldimethylammonium chloride) (PDADMAC) with poly(N-methyl-N-vinylformamide). They were able to obtain a stable 9 ACS Paragon Plus Environment

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multilayer film in pure aqueous solutions with this PSS and a random cationic copolymer with only 10 mol% PDADMAC by self-assembling 35.

Polymerization of DMAEMA. PDMAEMA was synthesized by a slightly modified ATRP protocol compared to previously reported work

36

(cf. Fig. 2; step a). For a well-predictable

reaction, initiation should be quantitative and immediate, and chain growth should be controlled. The polymerization was monitored by the measuring conversion of DMAEMA by 1

H-NMR spectra (Fig. S1). Those measurements have revealed that the reaction had an

almost linear increase of the conversion over a long time. The increase leveled off after 300 minutes, when the reaction was completed as far as possible. This steady growth rate enables the synthesis of precisely controlled polymers. The polymerization reached a conversion of 68% in 300 minutes. Since the conversion of DMAEMA should be proportional to the growth of the polymer chains if all initiator groups are actively participating in this reaction, the molar mass can be predicted from the conversion data obtained by 1H-NMR. This calculation lead to a molar mass of 15300 g/mol, which is very close to the results of SEC measurements with a number-average molar mass of 15700 g/mol. SEC measurement data are shown in SI, Fig. S2. The polydispersity was low, about 1.2, which also confirms the controlled character of the ATRP. The good agreement between the results for molar mass by the two different methods indicated that suited conditions for the controlled synthesis of the precursor polymer had been established.

Polymer-analogous conversions of PDMAEMA. The successfully synthesized precursor polymer PDMAEMA was converted in further steps with simple and efficient quaternization reactions to functional copolymers. First, the random copolymer PDMAEMA-co-PSPE was synthesized by the reaction of 1,3-propanesultone with tertiary amino groups of the PDMAEMA (cf. Fig. 2; step b). In order to be able to study the effects on LbL assembly, copolymers with three different fractions of the zwitterionic groups (0%, 25%, 50%) were targeted. The residual tertiary amino groups were quaternized with methyliodide to obtain a bifunctional copolymer with strong cationic anchor groups for electrostatically driven LbL assembly and zwitterionic groups for antifouling (cf. Fig. 2; step d). The structure of the resulting copolymer PSPE-co-PTMAEMA was determined by the precursor and experimentally confirmed by 1H-NMR and IR spectroscopy as well as elemental analysis. NMR and IR overview spectra are shown in SI, Figs. S3 to S7. The quaternization reactions were found to be very efficient in all the cases, and the degree of quaternization with 1,310 ACS Paragon Plus Environment

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propanesultone was well adjustable as observed by IR (Figure 3) as well as NMR and elemental analysis data (Table 1). wavenumber [cm-1] 900

950

1000

1050

1100

1150

1200

1250

1300

0 5 absorption [%]

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

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10 15 20 25 30 35 40 PTMAEMA-co-PSPE (0%)

PTMAEMA-co-PSPE (25%)


Figure 3: Sulfonate group and N-CH3 stretching regions of the IR spectrum of the copolymers PTMAEMA-coPSPE with varied fraction of zwitterionic groups.

For better comparison, the IR spectra of three polymers with varying fraction of zwitterions were superimposed and aligned in the absorption bands to obtain an identical baseline (cf. Figure 3). The signal of the ester group at 1720 cm-1 was used for that, because it was present in all three copolymers with the same intensity. The signals at 1035 cm-1 and 1170 cm-1 indicated an increasing fraction of zwitterionic groups, since these are specific for the sulfonate group of the sulfobetaine. It is clearly visible that both signals do not exist for PTMAEMA (0% PSPE) and systematically increase with increasing fraction of zwitterions. It was also observed that the signal at 950 cm-1 decreased with increasing fraction of zwitterions. This signal can be assigned to a characteristic C-N bond deformation which will disappear upon sulfobetainization. Hence, the IR spectra provide clear evidence for the fractional conversion to polycationic/-zwitterionic materials. Table 1: Degree of conversion to zwitterionic groups in PTMAEMA-co-PSPE and PTMAEMA-co-PSPE-coP(octylDMAEMA) (cf. Fig. 2), observed by 1H NMR and elemental analysis.

nominal zwitterion fraction 1

H-NMR spectroscopy elemental analysis

PTMAEMAco-PSPE (0%)* 0%

PTMAEMAco-PSPE (25%) 25%

PTMAEMAco-PSPE (50%) 50%

PTMAEMA-coPSPE (25%)+octyl** 25%

0% 0%

23.7% 23.3%

49.8% 48.4%

26.7 % 25.7 % 11

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* identical with PTMAEMA, i.e. simple polycation, used as reference ** identical with PTMAEMA-co-PSPE-co-P(octylDMAEMA); containing ~50% methyl and ~25% octyl groups introduced during step-wise quanternization

The copolymer with 25% zwitterionic fraction had also been quaternized with a combination of about 25% long octyl chains and 50% short methyl groups (cf. Fig. 2, steps c and d). The complete conversion of trialkyl amine into quaternary ammonium groups could be confirmed by 1H-NMR and FTIR spectroscopy. The degree of quaternization could be adjusted precisely as observed by 1H-NMR and elemental analysis (cf. Table 1). Overall, the different polymer-analogous reactions were performed successfully to yield well-defined functional polymers, with strong cationic anchor groups for electrostatically driven LbL assembly and zwitterionic groups for antifouling. And alternatively, they were also equipped with additional hydrophobic QAC groups as potential contact biocide.

3.2

LbL coating on silicon wafers

The main aim of this work had been the preparation of well-defined ultrathin multilayers which can stably and completely cover the substrate surface and impart novel surface functionalities. The polycation became due to the incorporation of zwitterionic groups a weakly interacting polymer. To counteract that issue, a "high" molecular mass, strongly interacting polyanion had been selected; i.e., PSS with M ~ 70000 g/mol was used. The different quaternization resulted in molecular masses from 17000 to 20300 g/mol. Only within a range of molecular masses up to 10000 g/mol, it was reported that atypical multilayering characteristics could occur during the LbL deposition 33,34. Nevertheless, Schlenoff et al. had also successfully used this PSS as polyanion to built a multilayer film with a “low” molecular mass polycation (M = 3500 g/mol) 37. The effect of the presence of the zwitterionic groups in the polycation was investigated first on silicon wafers as model substrates. The wafers were pre-modified with APTS to obtain an amino-functional polysiloxane monolayer to ensure the availability of positive charges under physiological conditions. The results of the LbL assembly on silicon wafer were analyzed in detail by ellipsometry, XPS, contact angle and zeta-potential measurements.


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18 total thickness [nm]

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

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16 14 12 10 8 6 4 2 0 0

1

2

layer

3

4

APTS

PTMAEMA (0%)



Figure 4: Added total layer thickness from ellipsometry measurements on pre-modified silicon wafers, coated via layer-by-layer assembly with PSS (blue framed) and PTMAEMA-co-PSPE with variation of zwitterionic group fraction (cf. Table 1) (red), as a function of the number of steps; samples measured after the final washing step of the respective layer deposition and drying of the coating under air blow.

Figure 4 is clearly demonstrating a systematic increase of the film thickness for all modification steps. Moreover, as for many LbL methods the thickness did not increase strictly linearly with the number of deposition cycles. The cationic layer was generally thicker than the anionic PSS layer. The polyelectrolyte multilayer thickness is generally affected by factors affecting the multi-film constitution like polymer concentration, salt type and concentration, pH value, deposition time, washing method, and temperature

33,38

, and by

the polyelectrolyte influences such as molecular weight and charge density of copolymers 35,39–41

. In this work, the adsorption conditions followed always exactly the same

experimental protocol, in particular low salt concentration at pH of 6 and room temperature. As reported by Seyrek et al. 34, the single polyelectrolyte layer had at small salt levels only a thickness of around 2 nm. This was also apparent in the ellipsometry data for the PSS layers (cf. Figure 4). On the other hand, for the polycation with 50% zwitterionic fraction which has the lowest charge density, the thickness difference between polyanion and polycation layer was generally most pronounced. Hoogeveen et al. have explained such behavior with regard to the charge overcompensation for polyelectrolyte multilayer growth

42,43

. Highly charged

polymers remained strongly bound to the surface and formed stable multilayers. By decreasing charge density of the adsorbing polyelectrolyte more polymer must adsorb to 13 ACS Paragon Plus Environment

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overcompensate and reverse the surface charge. Below a critical charge density the resulting surface charge does not allow the adsorption of polyelectrolytes of the next layer. The non linearity of the film thickness with the number of layer depositions indicates that different amounts of material were deposited in different steps. However, it should also be noted that the samples included an undefined amount of water because they were only dried by air flow and are hydrophilic due to the zwitterionic groups 44,45. The water content can be divided in "void water" which fills the empty space within the layers and "swelling" water which is directly bound to the polymer 41. The swelling ratios increase with increase of the polysulfobetaine content 46. The influence of the different degrees of “swelling” water in the last layer was also described by Schönhoff et al. to be the reason for the “odd-even effect” 47. An additional change in thickness was connected to replaceable "swelling" water, which after adsorption of the next layer ontop will be released as consequence of layer compression. Such behavior could also occur for the polycationic/zwitterionic building blocks because of their relatively low charge density (but not for PSS with its higher charge density) and may provide an alternative explanation for the observed non-linearity of thickness growth (cf. Figure 4). The thickness increase of the coatings made by polymers with zwitterionic fraction of 0% and 25% was similar, whereas the increase of the PSS layer was lower when using the polycation with 50% zwitterionic fraction. Analogous observations had also been made by Schoeler et al.

39

who had used the polyanionic PSS and random copolymers of

poly(diallyldimethylammonium chloride) (PDADMAC) and N-methyl-N-vinylacetamide with different percentages. There was a critical charge density between a percentage of 75% and 53% for the cationic PDADMAC. The authors had postulated that copolymers with low charge density adsorb on certain surfaces, but are easy desorbed when exposed to a solution with oppositely charged polymer. This could be one of the reasons that the polymer with 50% zwitterionic fraction did show less regular film growth. XPS is a surface-sensitive analysis technique which yields information about the atomic composition of the surface. To further confirm the LbL multilayer assembly on the silicon wafer, XPS spectra were analyzed with focus on peaks at binding energies of 103.3 eV for SiO2 (representing the substrate), 169.5 eV for S 2p, 285.5 eV for C 1s and 400.8 eV for N (NH3+, as indicator for the polycation). Results are shown in Fig. S8 and discussed in SI. Results confirm the coverage of the substrate and the incorporation of characteristic 14 ACS Paragon Plus Environment

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elements in the surface layer, which reveals the successful LbL assembly on the silicon surface. Contact angle measurements were also conducted to evaluate the hydrophilicity during the LbL assembly after deposition of each layer (Figure ). 70 60 contact angle [°]

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50 40 30 20 10 0 0

1

2 APTS

3

4 layer

5

6

PSS (0%)


PSS (25%)


PSS (50%)


7

8

Figure 5: Water contact angle (sessile drop and Young Laplace fit) of pre-modified silicon wafer coated via LbL deposition with PSS (blue) and PTMAEMA-co-PSPE with variation of zwitterionic fraction (cf. Table 1) (red), as a function of the number of deposition steps.

The results for the step-wise coating of the silicon wafer illustrate the alternating deposition of PSS and the different PTMAEMA-co-PSPE layers, which suggest a well-defined multilayer system. The APTS-modified silicon wafer had a water contact angle of about 56°

29

. The

alternating deposition of the polyelectrolytes led to a decrease in the contact angle for up to six layers. Additional layers did not lead to any further changes for the respective layer. The contact angle was generally lower after deposition of PSS than after PTMAEMA-co-PSPE. However, a zwitterionic group-containing layer should be formed on the outer surface of the coated material; therefore the contact angles after PTMAEMA-co-PSPE deposition are more relevant. When the polymer without zwitterionic fraction was used as polycation, contact angles of about 40° were reached. After adding zwitterions to the polycation (25% and 50%) the surface had much lower contact angle, down to 22°. Such large decrease in contact angle caused by zwitterions has been reported regularly and is attributed to their electrostatic interactions with water which enables the formation of a stable hydration layer

48

. After

deposition of the 6th layer, no significant difference between 25% and 50% zwitterionic 15 ACS Paragon Plus Environment

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fraction had been found. Also, the oscillatory change of contact angle for the alternating layers seen for PTMAEMA and PSS was not observed for PTMAEMA-co-PSPE and PSS between 6th and 8th layers. This can also be attributed to more efficient hydration layer formation. However, the zetapotential data revealed a very clear difference between surfaces after six layer depositions as function of zwitterions fraction; only with PTMAEMAco-PSPE of 50% zwitterion fraction, a close to neutral surface in the pH range between 3 and 9 had been obtained (see SI, Figure S9; cf. also Figure 6a, below). This is especially relevant because strongly hydrophilic and neutral surfaces will have antifouling properties.

3.3

LbL coating of PDMS (catheter material)

After the feasibility of the coating had been demonstrated for the model silicon wafers, the next step was to transfer the method to the catheter material PDMS. Therefore the samples were also pre-functionalized with APTS, here after controlled hydrolysis of the silicone material surface. The presence of primary amino groups on thus treated samples was confirmed by the ninhydrin test. The alternating deposition of polyelectrolyte layers was then done under the same conditions as established for the silicon wafers. The contact angles of the PDMS surface and the materials after the different modification steps were evaluated with the captive bubble method and are shown in SI, Figure S10. During the layer-by-layer assembly, the contact angle decreased to 22° just like the contact angle of the silicon wafers modified with six layers. However, in contrast to the silicon wafers the contact angle of the catheter material indicated only a weak oscillating change between the alternating layers. Furthermore, the contact angle data for the different polycations were relatively similar. Even through the addition of 25% hydrophobic octyl groups to the polycationic PTMAEMA-co-PSPE 25% in the sixth layer only a very minor increase of the contact angle was observed. Overall, contact angle data demonstrated that the surface was successfully modified via LbL deposition and became hydrophilic. That an effect of the zwitterionic fraction in the polycation on contact angle could not be observed may also be because of the morphological/structural features of PDMS. Indeed, the AFM analyses showed a structure of the surface with some regular macroscopic features (cf. Figure below). Furthermore, stability tests were performed in aqueous solutions of sodium azide (10 mM) as well as in synthetic urine and were analyzed by contact angle measurements (SI; Figure 16 ACS Paragon Plus Environment

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S11 and S12). The contact angle of the modified catheter material remained unchanged; i.e. the hydrophilicity of the modified PDMS was not influenced by the exposure to water or synthetic urine for up to 10 days. The contact angle of the unmodified PDMS decreased, but the constant values reached after 10 days were still much larger than for the LbL coated materials. This effect could result from migration of the filler silica to the PDMS surface. It is well-known that the polysiloxane matrix can rearrange due to the low glass transition temperature in order to minimize interfacial energy 49,50. In contact with an aqueous phase, the polysiloxane silica composite will thus result in a more hydrophilic surface compared to the native dry state. Overall, LbL assembly of polyelectrolytes on pre-modified PDMS surfaces seems to be a good strategy to functionalize that catheter material (for an analogous approach, but using different polyelectrolytes see 51). The surface charge of the PDMS catheter material was investigated by zeta potential measurements after each coating step. In Figure , the dependency of zeta potential at pH 6.5 for the six-layer coating with the variation of zwitterions fraction in the polycation is shown for different materials, i.e. the silicon wafer (Figure 6a) and the PDMS catheter material (Figure 6b). The observed oscillating zeta potential changes in all cases could be attributed to the alternative immersion in either positively charged or negatively charged polyelectrolyte solutions; the odd numbered layers had negative and the even numbered layers had positive zeta potential. These results are characteristic for electrostatically driven LbL assembly and indicated a well-defined, step-wise deposition of the negatively charged PSS and the positively charged PTMAEMA-co-PSPE.


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Figure 6: Zeta potential values at pH 6.5 of APTS pre-modified Si wafer (a) and of APTS pre-modified PDMS catheter material (b-d) during LbL deposition toward a six-layer coating with polycations of varied zwitterionic fraction (cf. Table 1) (red) and the polyanion PSS (marked in blue; b), or also containing the enzyme CDH in either layer #3 (c) or layer #5 (d).

The zeta potential data were almost identical for the PSS layers. However, the values for the cationic layers were different; the absolute value decreased, i.e. the surface became more neutral, with increasing zwitterionic content in the polycation. Analogous to the contact angle data, the influence of the different polymers was more apparent on the smooth silicon wafer than on the PDMS catheter material. In the investigation of the silicon wafers the zeta potential of the cationic layers diverged more from each other with increasing layer numbers. For the 6th layer the difference between the highest and the lowest value, i.e. the effect of 50% zwitterionic fraction, was 53 mV. This effect was less visible for the coated catheter material. However, when comparing zeta potential for the entire pH range from ~3 to ~10, the effect of the varied zwitterions fraction was very well visible for both silicon wafer and silicone material (for complete data see SI, Figures S9 and S13). The zeta potential data for 0% zwitterionic fraction were all in the positive range, thus no isoelectric point could be detected. An increasing fraction of zwitterions in the polycation decreased the isoelectric point; at 50% zwitterion fraction the isoelectric point was pH value ~7.5. Yang and Ulbricht 18 ACS Paragon Plus Environment

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had shown that the zeta potential of a surface which is fully covered by the polysulfobetain PSPE is in the negative range but very close to zero, i.e. the surface is almost zero

52

. The

reason for the effective charge neutralization in this particular polyzwitterion had been attributed to the very effective intra-side chain ion-pairing because of the similar charge density at the sulfonic acid and quaternary ammonium groups 53. Hence, for all coatings and throughout the entire pH range the effect of the positively charged PTMAEMA segments in the polycation/zwitterions could be seen, but its influence was largely reduced by the zwitterionic segments yielding a close to neutral outer surface when using PTMAEMA-coPSPE with 50% PSPE fraction for LbL coating. Through the quaternization of 25% of PTMAEMA-co-PSPE with 25% zwitterions fraction with octyl groups, the isoelectric point shifted slightly into basic region, to a pH value of ~ 10 (cf. SI, Figure S13). In experiments shown in Figure c and 6d, an enzyme layer was additionally incorporated in the coating. Due to its low isoelectric point (at pH 4.2 54), the enzyme CDH was assembled as polyanion instead of PSS as the 3rd or 5th layer. The results of the zeta potential measurements for the 6th layer of the coating with the integrated enzyme were similar to the ones without enzyme. Only the enzyme layers had a different zetapotential compared to the other anionic (PSS) layers; at a pH value of ~ 6.5 a neutral surface was observed. This is higher compared to the isoelectric point for CDH of pH 4.2, but can be well explained: Due to its amphoteric character and relatively fixed charge distribution over its surface, the enzyme had probably been embedded in a such conformation that more negative charges had been oriented to the cationic layer below, leaving more positive charges free thus causing the isoelectric point of this layer to be significantly higher than for the free enzyme. The enzyme immobilization was also analyzed by contact angle measurements (see SI, Figure S14). During the LbL deposition the surfaces became more hydrophilic and there was also no clear relation to the varied zwitterionic fraction of the exposed polymer. However, this indicated that all systems developed a well defined multilayer architecture that is based on electrostatic attractive forces, even if certain building blocks do not have strong net charge like the enzyme CDH or the polycation with 50% zwitterionic fraction. The changes of surface morphology of the multilayered coatings were visualized by AFM. Clear differences between unmodified base and modified PDMS materials as well as specific effects of different drying methods were found (see Figure , and Figs. S15-17 and Table S1).


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Figure 7: AFM topography images of air dried, unmodified catheter material (PDMS) (a), APTS pre-modified air dried PDMS (b), air dried PDMS with a six-layer coating containing the polycations PTMAEMA (0%) (c), or PTMAEMA-co-PSPE (50%) (d); influence of drying process onto six-layer coated PDMS with PTMAEMA-co-PSPE (50%): under water (e) and freeze dried (f).

The base material, shown in Figure a, presented a relatively smooth, compact and microscopically homogeneous surface with some regular macroscopic features due to the preparation process of solvent casting. In comparison to topography of the native PDMS, the APTS-functionalized PDMS clearly showed increased roughness, i.e. an interesting tree-like topography on top of the PDMS. A reason for this structure could be the alignment tendency of the APTS-based polysiloxane after the drying process 55. The particular structure seems to have been formed upon drying, and the appearance suggests that the amino-functional layer had been partially formed by condensation and the chemical grafting density on the PDMS surface may be relatively low. PDMS is well known for its surface rearrangement after hydrophilic modification, due to its intrinsic hydrophobicity and low glass transition temperature 49. This reconstruction of the surface is a problem in some modification processes and it can also enhance the formation of the alignment structure of the APTS layer during drying process. One effective method for PDMS surface coating is layer-by-layer assembly. As shown in Figure c and 7d, the surface was fully and much more regularly 20 ACS Paragon Plus Environment

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covered after the modification with a six-layer LbL coated system; the topography images presented a uniform structure. This significant change of the topographies indicated a total coverage of the PDMS surface. However, it made a large difference with respect to surface topography, whether zwitterionic segments were inside the multilayer assembly: For the sample without zwitterions in the polycation the coverage seemed to be smoother than for the one with 50% zwitterion fraction. The presence of the relatively regular macroscopic pattern, especially for 50% zwitterion fraction, indicated segregation on the surface. The polymer with 50% zwitterions had much less cationic groups, which will lead to a less interacting multilayer assembly; less inter-connection is formed, and during the drying process, a higher degree of segregation occurred. The domains were larger for the polycation with 50% zwitterion fraction because of less confinement of the polymer layer. This can be linked to the low charge density. Weakly charged polyelectrolytes tend to adsorb in conglomerate conformation with a larger amount than highly charged polyelectrolytes; when the surface charge density is too low to spread a chain over the surface, the loss in configurational energy is compensated by segmental population of loops 56. During the deswelling process of the APTS layer the coating is reordered in a new structure and all subsequent layers adopt this conformation what leads to segregation. This tendency is increased by the entangled structure with loops in the weak charge density case. It must also be taken into account that the systems presented here consist only of relatively few layers (that decision had been taken because the change of surface properties had leveled off /cf. Fig. 5/ and a small number of coating steps is attractive from application point of view), and they are thus in a regime where the layer properties could still be dominated by the substrate

57

. Dewetting of the PDMS surface could be considered as driving force, and this

may be caused by chemical and physical properties of the substrate

57

. According to the

contact angle data (cf. Fig. 5), the APTS primed surface has not much different wetting properties compared to the ones of the added layers. However, as already evoked above, the microscopic chemical heterogeneity and dynamics of the APTS layer must be considered. The possible physical scenario for dewetting, caused by polyelectrolyte induced space charge in the substrate and resulting charge repulsion 57, is not probable in this case because of the low dielectric constant of PDMS. Another evidence for an influence of the drying method could be taken from the comparison with the not-dried samples. Figure 7e presents the AFM image under water of a six layer 21 ACS Paragon Plus Environment

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system with 50% fraction of zwitterions in the polycation, indicating that the surface was much more densely covered; degree of segregation seemed to be significantly smaller compared to dry samples. After 1 hour re-equilibration the air-dried sample in water, the polymer on the surface swelled and formed a small ring-like structure as shown in the Supporting Information (Fig. S17). This topography was different from the one for the never dried material (cf. Figure 7e). Hence, it seems that layer segregation upon drying can be reduced but not completely eliminated. To enhance the stability of multilayer assembly the layers could be cross-linked to each other 58. It is commonly assumed in literature

59,60

antifouling abilities. Vrijenhoek et al.

, that surfaces with lower roughness have better

60

demonstrated a correlation between surface

roughness and fouling with respect to the flux through membranes. It had been proposed that the foulants are likely to be absorbed in the valleys of the rough surface. To decrease the extent of fouling it would be important to generate smoother surfaces which can restrain the adsorption and deposition of foulants. To prevent this behavior during storage, further drying methods such as freeze-drying were evaluated. Indeed, Figure f shows the smoother surface and the less intensely developed segregation after freeze-drying. Overall, the results obtained with all different characterization methods revealed that the critical charge density limit for electrostatic interactions which allows multilayer formation has not been reached with 50% zwitterionic fraction. In addition, the enzyme was firmly embedded without disturbing the layer sequence. Considering the above discussion of the segregation tendency of the LbL layers built with polycation/zwitterions, it must be taken into account that the location of the enzyme may not be as well defined as indicated in Figure 1. However, an effective immobilization can be expected and especially the freezedrying procedure is expected to preserve both enzyme localization and activity. Finally, the outer layer was a polycation, preferably with a higher zwitterionic fraction for good antifouling effect.

3.4

Biological activity of LbL coated PDMS (catheter material)

Antimicrobial and antibiofilm properties of the coated samples were determined by measurements of enzyme activity and biofilm formation as well as by confocal laser scanning microscopy (CLSM) visualization. These tests were carried out with samples which had either not been dried or been freeze-dried, because of the surface segregation effects shown in 22 ACS Paragon Plus Environment

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section 3.3. The results of the enzyme activity measurements are summarized in Figure and show that all PDMS samples which had CDH deposited in its multilayer coating were able to produce H2O2. 4.5 4.0 H2O2 production [nmol/ cm² h]

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3.5 3.0 freeze dried - 3CDH

2.5

freeze dried - 5CDH

2.0

not dried - 3CDH 1.5

not dried - 5CDH

1.0 0.5 0.0 base material

0%

25%

50%

octyl

Figure 8: H2O2 production after oxidation of cellobiose, catalyzed by CDH, for untreated catheter material in comparison to six-layer coated materials containing CDH in either the third or the fifth layer and built up using polycations with zwitterionic group fractions of 0%, 25% , 50% and 25% plus octyl groups (cf. Table 1); data obtained from Amplex red assay.

This study clearly demonstrated that the different storage methods, either in 20 mM citrate buffer (pH 5.5) or freeze dried, the position of CDH in the multilayer system, either in layer #3 or #5, as well as the fraction of zwitterions present in the polycation had large influences on the activity of the immobilized CDH. Freeze dried samples showed a two to three fold higher H2O2 production capacity when compared to the samples stored in buffer, which is probably due to the fact that immobilized enzymes tend to lose activity when stored in solution. When CDH, acting as polyanion, was embedded in the third layer the enzyme activity was drastically reduced as compared to the samples with the enzyme in the fifth layer. The additional layers of alternating polyanion and polycation seem to shield the enzyme and to hamper the diffusion of substrate to the enzyme as well as the release of the product out of the multilayer system. The third factor influencing the enzyme activity is the fraction of zwitterion in the polycation layer. Increase of the fraction leads to a significant decrease in enzymatic activity. The charge density, which varies with the fraction of zwitterions, has apparently affected the interaction between enzyme and polyelectrolyte. It 23 ACS Paragon Plus Environment

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seems that the highest positively charge density polycation (modification without zwitterion) built the most stable alternating multilayer with the largest amount of enzyme activity. That the effect of enzyme location onto activity can been found also for not dried samples when using polycations with 25% PSPE fraction (including the octyl version; cf. Fig. 8) indicates that the assemblies may at least partially preserve a vertically segregated structure with respect to the enzyme as indicated in Figure 1. Since the effect of polyzwitterion fraction is more pronounced in the freeze-dried samples, the drying effect on layer structure could also play a role, as discussed section 3.3. During segregation of the coating, the enzyme was probably more firmly embedded in the assembly which could also hinder diffusion of the substrate. To summarize, it can be stated that the different samples, especially the ones which were freeze dried, produced H2O2 in a range of 2.4 - 3.9 nmol/cm²h which had proven to have an antimicrobial effect in previous studies 61. The results show that it was possible to immobilize enzymes by this method, and the freeze dried samples with CDH in the fifth layer achieved very good results for the activity of the enzyme. 120 100 biofilm growth [%]

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80 freeze dried - 3CDH 60

freeze dried - 5CDH not dried - 3CDH

40

not dried - 5CDH 20 0 base material

0%

25%

50%

octyl

Figure 9: Colorimetric (crystal violet) assay for quantification of Staphylococcus aureus biofilm formation on catheter material (PDMS) after different LbL coatings with six layers containing CDH in either layer #3 or #5 and using polycations with zwitterionic group fractions of 0%, 25%, 50% and 25% plus octyl groups (cf. Table 1).

The prevention of bacterial biofilm formation was assessed via crystal violet staining to quantify the biofilm biomass deposited on the PDMS surface. In Figure , biofilm growth was calculated from data of absorbance at 595 nm which are shown in SI, Figure S18. It could be observed that the functionalized PDMS samples reduced the amount of biofilm development 24 ACS Paragon Plus Environment

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in contrast to unmodified PDMS material. The freeze-dried samples showed biofilm reduction in the range of 20-40% whereas the samples stored in citrate buffer, reduced the amount of total biomass in a range of 37-53%, all when compared to the base material. The better prevention of biofilm formation of the buffer stored samples could originate from their smoother surface, because the segregation of the dried samples was not completely reversible after re-immersion in water (cf. section 3.3). There was no clear trend for reduction of attachment of bacteria in response to the variation of the concentration of zwitterions or octyl moieties in the polycation. This, however, means also that the octyl substitution had no additional beneficial effect on this parameter. To evaluate the efficiency of the coating to inhibit biofilm formation the samples were analyzed with CLSM of LIVE/DEAD stained Staphylococcus aureus biofilms. Figure shows the distribution of live and dead cells directly on the surface after fluorescent staining of the bacteria.

Figure 5: CLSM images of catheter material (PDMS) after different LbL coatings with six layers containing CDH in either layer #3 or #5 and using polycations with zwitterionic group fractions of 0% (b), 25% (c), 50% (d) and 25% plus octyl groups (e). Row (a) shows pristine catheter material, which served as reference.

The uncoated base PDMS had a very uniform distribution of individual colonies which are fairly close together. Compared to this the amount of cells attached to the coated PDMS 25 ACS Paragon Plus Environment

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samples was in most cases drastically reduced. An effect of storage method was recognized similar to the quantification data (cf. Figure 9). The freeze dried samples showed little clusters of live (green) cells and quite a lot of dead bacteria (red). Better antifouling effects of the modification were achieved for the coated PDMS samples which were stored in citrate buffer. The fluorescence images of the not dried samples only show individual isolated growth of bacteria. On the other hand, the higher H2O2 production by the freeze dried samples (cf. Figure 8) seems to have a positive effect as a higher amount of dead bacteria could be found on the surface. Quite a lot of dead bacteria were found on the PDMS surfaces which are modified by using polycations with zwitterionic and octyl groups. This could be achieved through the introducing of QAC groups.

4. Conclusions Layer-by-layer assembly was successfully used to deposit multifunctional polymers comprising zwitterions and quaternary ammonium groups and to immobilize enzyme to construct a new kind of anti-biofouling and antimicrobial coating for urinary catheters. In the synthesis part of the work, it was shown that the polycation PTMAEMA can be successfully combined with specified fractions of polyzwitterion (PSPE) and octyl-ammonium functional QAC. These random copolymers of targeted charge density have been adsorbed in an alternating manner with the strongly charged polyanion PSS. Contact angle, XPS, ellipsometry, zeta-potential and AFM measurements provide evidence that it is possible to prepare zwitterionic multilayers via LbL assembly on different substrates such as silicon wafers and PDMS tube and balloon catheter materials. The observation of absence of systematic changes in the contact angle and the attenuated growth in ellipsometry when using the polycation with 50% zwitterions indicated deviations from strictly regular growth due to reduced charge density. However, the combination of all data provides strong evidence that with up to 50% zwitterion fraction in the polycation the critical charge density limit which is needed to obtain significant layer growth has not yet been reached. All surfaces remained stable for up to 10 days in water and in synthetic urine. By increasing the zwitterionic fraction, surfaces became more neutral and hydrophilic. The charge density had a pronounced effect on the drying process as well. Lower charge density caused less interaction between the layers in the multilayer system, which resulted in more segregation within the layer system during the drying process, especially by simple air drying. This 26 ACS Paragon Plus Environment

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resulting roughness of the surface can be reduced by freeze drying. This multilayer system was also successful used as reservoir for enzyme immobilization. CDH which is able to produce H2O2 in the presence of polysaccharides was effectively embedded in the multilayer system and could preserve its enzyme activity. For that it was shown that the layer architecture, i.e. the location of the enzyme in the layer, has an influence on the activity of enzymes; this could be linked to substrate diffusion hindrance. The CDH functionalized catheter material exhibited more antimicrobial activity after freeze drying. An additional antibacterial effect could be obtained by introducing QAC groups. Based on the biological results presented that the multilayer films can not only reduce the bacterial adhesion down to 40% relative to uncoated PDMS but also kill the bacteria adhered onto the surface. Therefore, the established method toward multilayers with immobilized CDH and zwitterions with additional QAC groups as contact biocides could be a viable alternative for antibiofouling and antimicrobial coatings on urinary catheters.

Acknowledgments. This research was performed in the framework of the European project NOVO (FP7-27840) “Novel approaches for prevention of biofilms formed on medical indwelling devices, e.g. catheters”. The authors thank Dr. Steffen Franzka (Interdisciplinary Center for Analytics on the Nanoscale /ICAN/, Universität Duisburg-Essen /UDE/) for the AFM measurements as well as Marc Thomas (Technische Chemie II, UDE) and Dr. Ulrich Hagemann (ICAN, UDE) for the XPS measurements. This work has also been supported by the Federal Ministry of Science, Research and Economy (BMWFW), the Federal Ministry of Traffic, Innovation and Technology (bmvit), the Styrian Business Promotion Agency SFG, the Standortagentur Tirol, and the Government of Lower Austria and Business Agency Vienna through the COMET-Funding Program managed by the Austrian Research Promotion Agency FFG.

Supporting Information Available. Reaction control of the ATRP synthesis; Details of polymer characterization methods; SEC analysis of PDMAEMA; PDMAEMA,

PDMAEMA-co-PSPE,

PTMAEMA-co-PSPE,

1

H-NMR spectra of

PTMAEMA-co-PSPE-co-

P(octylDMAEMA); IR spectra of PTMAEMA-co-PSPE; Details of physical and physico-chemical methods for characterization of the coatings; XPS analyses and their discussion; zeta 27 ACS Paragon Plus Environment

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potential data from pH 3-10 for the various modifications; contact angle measurements of PDMS and for samples after the various modifications, including stability tests; further AFM topography data; details of methods for functional characterization of the coatings; absorbance data for crystal violet assay. This material is available free of charge via the internet.

References (1)

Siddiq, D. M.; Darouiche, R. O. New Strategies to Prevent Catheter-Associated Urinary Tract Infections. Nat. Rev. Urol. 2012, 9 (6), 305–314.

(2)

Topal, J.; Conklin, S.; Camp, K.; Morris, V.; Balcezak, T.; Herbert, P. Prevention of Nosocomial Catheter-Associated Urinary Tract Infections through Computerized Feedback to Physicians and a Nurse-Directed Protocol. Am. J. Med. Qual. 2005, 20 (3), 121–126.

(3)

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