A SIMULTANEOUSLY ANTIMICROBIAL AND PROTEIN-REPELLENT POLYZWITTERION

Abstract

The present invention concerns a simultaneously antimicrobial and antifouling and protein repellent polyzwitterion (monolayers, polymer networks and surface-attached polymer networks formed thereby), and substrates coated with the inventive simultaneously antimicrobial and antifouling and protein repellent polyzwitterion. The invention also concerns uses of the inventive polymers and substrates for preventing and combating microbial growth.

Claims

1.-19. (canceled)

20. Antimicrobial and antifouling polymer comprising a molecular weight of more than 5,000 g mol.sup.1 and as a repeat unit a structure according to formula (I): ##STR00020## wherein X is O; Y is selected from hydrogen or is a negative charge; A is O; Z is (CH.sub.2).sub.qN.sup.+(R.sub.3R.sub.4R.sub.5), wherein: R.sub.3, R.sub.4, R.sub.5 are independently from each other selected from either H or an C.sub.1-C.sub.6-alkyl; q is an integer selected from a range of 1 or 3 to 10; or Z is (CH.sub.2).sub.qN(R.sup.11R.sup.12), wherein: R.sup.11, R.sup.12 are independently from each other selected from either H or an C.sub.1-C.sub.6-alkyl; q is an integer selected from a range of 1 or 3 to 10; and n is an integer selected from a range of 10 to 2500.

21. Antimicrobial and antifouling polymer comprising a molecular weight of more than 5,000 g mol.sup.1 and as a repeat unit a structure according to formula (I): ##STR00021## wherein X is O; Y is selected from hydrogen or is a negative charge; A is NH; Z is (CH.sub.2).sub.qN.sup.+(R.sub.3R.sub.4R.sub.5), wherein: R.sub.3, R.sub.4, R.sub.5 are independently from each other selected from either H or ethyl-, propyl-, or isopropyl-; q is an integer selected from a range of 1 to 10; or Z is (CH.sub.2).sub.qN(R.sup.11R.sup.12), wherein: R.sup.11, R.sup.12 are independently from each other selected from either H or ethyl-, propyl-, or isopropyl-; q is an integer selected from a range of 1 to 10; and n is an integer selected from a range of 10 to 2500.

22. Antimicrobial and antifouling polymer comprising a molecular weight of more than 5,000 g mol.sup.1 and as a repeat unit a structure according to formula (I): ##STR00022## X is CR.sub.1R.sub.2, wherein: R.sub.1 and R.sub.2 are independently from each other selected from linear or branched C.sub.1-C.sub.6 alkyl, Y is selected from hydrogen or is a negative charge; A is NH; Z is (CH.sub.2).sub.qN.sup.+(R.sub.3R.sub.4R.sub.5), wherein: R.sub.3, R.sub.4, R.sub.5 are independently from each other selected from either H or an C.sub.1-C.sub.6-alkyl; q is an integer selected from a range of 1 to 10; or Z is (CH.sub.2).sub.qN(R.sup.11R.sup.12), wherein: R.sup.11, R.sup.12 are independently from each other selected from either H or an C.sub.1-C.sub.6-alkyl; q is an integer selected from a range of 1 to 10; and n is an integer selected from a range of 10 to 2500.

23. Antimicrobial and antifouling polymer according to claim 22, wherein the repeat unit with a structure according to formula (I) is selected from any of formulae (Ib) or (Ie): ##STR00023## wherein: R.sub.1 and R.sub.2 are independently from each other selected from linear or branched C.sub.1-C.sub.6 alkyl,

24. Antimicrobial and antifouling polymer comprising a molecular weight of more than 5,000 g mol.sup.1 and as a repeat unit a structure according to formula (I): ##STR00024## wherein X is CCR.sub.1R.sub.2, wherein: R.sub.1 and R.sub.2 are independently from each other selected from hydrogen (H), linear or branched C.sub.1-C.sub.6 alkyl, Y is selected from hydrogen or is a negative charge; A is NH or O; Z is (CH.sub.2).sub.qN.sup.+(R.sub.3R.sub.4R.sub.5), wherein: R.sub.3, R.sub.4, R.sub.5 are independently from each other selected from either H or an C.sub.1-C.sub.6-alkyl; q is an integer selected from a range of 1 to 10; or Z is (CH.sub.2).sub.qN(R.sup.11R.sup.12), wherein: R.sup.11, R.sup.12 are independently from each other selected from either H or an C.sub.1-C.sub.6-alkyl; q is an integer selected from a range of 1 to 10; and n is an integer selected from a range of 10 to 2500.

25. Antimicrobial and antifouling polymer according to claim 24, wherein the repeat unit with a structure according to formula (I) is selected from any of formulae (Ic) or (If): ##STR00025##

26. Antimicrobial and antibiofouling polymer according to claim 20, wherein the polymer comprises a molecular weight M.sub.n of between 5,000 g mol.sup.1 and 1,000,000 g mol.sup.1, preferably between 10,000 g mol.sup.1 and 500,000 g mol.sup.1, more preferably between 20,000 g mol.sup.1 and 500,000 g mol.sup.1, and even more preferably between 20,000 g mol.sup.1 and 200,000 g mol.sup.1, between 20,000 g mol.sup.1 and 150,000 g mol.sup.1, or between 20,000 g mol.sup.1 and 100,000 g mol.sup.1, most preferably between 20,000 g mol.sup.1 and 95,000 g mol.sup.1.

27. Antimicrobial and antibiofouling polymer according to claim 20, wherein 0.05% to 10 wt % of the Z groups of the antimicrobial and antibiofouling polymer of formula (I) are replaced by a crosslinking unit.

28. Polymeric network comprising an antimicrobial and antifouling polymer according to claim 20 and a crosslinker, wherein the polymer network is formed by crosslinking the antimicrobial and antifouling polymers with a crosslinker.

29. Polymeric network according to claim 28, wherein the crosslinker comprises a photo-crosslinking unit or a thermo-crosslinking unit.

30. Substrate comprising an antimicrobial and antifouling polymer according to claim 20, wherein the antimicrobial or antibiofouling polymer is attached covalently or non-covalently onto said substrate.

31. Substrate according to claim 30, wherein the surface attachment occurs via a molecule for surface attachment comprising a photo-crosslinking unit or a thermo-crosslinking unit.

32. Substrate according to claim 30, wherein the polymeric layer formed on the substrate by the antimicrobial or antifouling polymers has a thickness of about 10 nm to about 1000 m.

33. Use of an antimicrobial and antifouling polymer according to claim 20 for preventing microbial growth and biofouling on a substrate, device or tool.

34. Use according to claim 33, wherein the surface of the substrate is an organic or an inorganic surface.

35. Use according to claim 34 wherein the inorganic surface is selected from the group consisting of surfaces comprising metals or alloys, silicon surfaces, or ceramic surfaces.

36. Use according to claim 34, wherein the organic surface is selected from polymeric surfaces including oxidized poly(styrene), oxidized poly(ethylene), (substituted) poly(ethyleneimine) (PEI), (substituted) poly(vinylpyridine) (PVP), (substituted) PVP-based polymers and co-polymers, poly(diallyldimethylammonium)-based, (substituted) poly(butylmethacrylate-co-amino-ethyl methyl-acrylate), (substituted) poly(2-(dimethyl-amino)-ethyl methacrylate)-based surfaces, co-polymers thereof, fluorinated polymers or co-polymers thereof, silicone polymers or co-polymers thereof, or any further polymer suitable for such an approach.

37. Use according to claim 33, wherein the substrate is selected from the group consisting of an implant, a prosthesis, a joint, a bone, a tooth, screws, anchors, fastener or fixing material, a medical or surgical device or tool, implant trephine or trepan drill, scalpels, forceps, scissors, screws, fasteners and/or fixing material used for implantation, holders, clips, clamps, needles, linings, tubes, water tubes, pipes, water pipes, bottles and bottle inlays, breathing hoses, inlays for medical equipment, surfaces of operating tables, treatment chairs, catheter, stents, any wound dressing material, including plaster, gazes, bandages, bed sheets for clinical or medical purposes, sheets for covering medical devices, book covers, keyboards, computer keyboards, computer, laptops, screens, displays, display covers, lamps, grips of tools and instruments, a biomaterial suitable for tissue support, a tissue carrier system for wound dressing or for volume preservation of solid body tissues, substrates used for storage of cells, tissues, or organs, substrates used for storage of food, refrigerators, coolers, and storage boxes.

38. Antimicrobial and antifouling polymer according to claim 20 for use in treating or preventing microbial infections in a patient.

Description

FIGURES

[0154] The figures shown in the following are merely illustrative and shall describe the present invention in a further way. These figures shall not be construed to limit the present invention thereto.

[0155] FIG. 1: illustrates the structure and synthesis of the inventive antimicrobial and antifouling polymer, a carboxybetain-based polyzwitterion (PZI, 1), as shown in FIG. 1. PZI 1 is obtained via a protective group approach. The PZI monomer precursor 2 carries a tert-butoxy carbamate (Boc) protective group on the primary amine. It is obtained in a one-step reaction from oxonorbornene-anhydride 3 and N-Boc-ethanolamine 4. Polymerization by Grubbs IIIrd generation catalyst (G3) in dichloromethane yields the polymer in its protected form (5). Gel permeation chromatography (in THF) showed that high molecular weights up to 73,000 g mol.sup.1, with a polydispersity index of 1.5 g mol.sup.1, were easily available. The Boc protective group was removed by hydrochloric acid in dioxane, giving the desired PZI 1.

[0156] FIG. 2: illustrates in the top section exemplarily how PZI coated surfaces (6) were obtained by applying PZI 1 and the external crosslinker 7 (carrying thiol groups as photo-crosslinking units) to a surface 8 that has been pretreated by a molecule for surface attachment carrying benzophenone as a photo-crosslinking unit and a silane as a reactive group. UV irradiation triggers two UV-activated reactions simultaneously: the CH-insertion reaction between aliphatic CH groups of the PZI 1 and keto groups of a benzophenone-pretreated substrate 8 (which causes covalent attachment between the PZI and the molecule for surface attachment) and the thiol-ene reaction between the PZI double bonds and the thiol groups of the external crosslinker 7, which causes network formation. Overall, a surface-attached polymer network is thus obtained. The process is not limited to standard laboratory surfaces like glass, silicon wafers or quartz, but works on many technical surfaces that carry OH groups or can be oxidized by simple plasma cleaning. This includes most medical plastics and many medically important metals including titanium and aluminum. [0157] The process as described in FIG. 2 works with the deprotected PZI 1, or its protected version 6. [0158] FIG. 2, shows in the bottom section 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 Coumarin-labeled PZI used).

[0159] FIG. 3: illustrates the assessment of the antimicrobial activity of the PZI coated surface 6 (see also FIG. 2) compared to two reference surfaces, the strongly antimicrobial butyl SMAMP (as described in P. Zou, D. Laird, E. K. Riga, Z. Deng, H.-R. Perez-Hernandez, D. L. Guevara-Solarte, T. Steinberg, A. Al-Ahmad, K. Lienkamp, Journal of Materials Chemistry B 2015, 3, 6224-6238) and the strongly protein-repellent PSB network (as described in S. Colak, G. N. Tew, Langmuir 2012, 28, 666-675). These reference polymers were immobilized as surface-attached networks using the same process as described above for the PZI. By comparing the PZI to these structurally similar materials which either had only an antimicrobial effect (i.e. intrinsic antimicrobial activity), or only an anti-adhesive effect, but no intrinsic antimicrobial activity, the unique properties of the inventive PZI can be appreciated. [0160] This figure shows time-kill experiments on the PZI 6, the SMAMP and the PSB networks. The experiments were performed using standardized procedures. In short, the test surfaces were sprayed with either Gram negative E. coli bacteria, or Gram positive S. aureus bacteria (at 10.sup.6 bacteria per cm.sup.3). After different incubation times on the test surfaces, the surviving bacteria were plated out on agar plates, where they were further incubated, and counted. In this figure, the number of colony forming units, in percent, relative to the negative control, is plotted versus incubation time. Thus, this assay quantifies the relative amount of viable bacteria after a certain exposure to the surface. The data indicates that both PZI and SMAMP were highly active against S. aureus (a). Surprisingly, the PZI killed S. aureus faster and more quantitatively than the strongly antimicrobial SMAMP. While there was a residual growth of 3.7% for the SMAMP after 4 h, the growth after 4 hours PZI exposure was 0.0% (i.e. at the detection limit of the method). Both surfaces quantitatively killed E. coli bacteria (b, 0.0% growth after 4 h, FIG. 3b.). The protein-repellent PSB had growth rates >100% for all time points studied and was thus, as predicted, not antimicrobial.

[0161] FIG. 4: Shows the results of biofilm formation experiments. In these biofilm formation experiments, E. coli and S. aureus bacteria were grown on the test substrates over 72 hours in the presence of nutrients. After defined time points, the growing biofilm was stained using the BacLight Bacterial Viability Kit. This kit stains membrane-compromised cells red, while all cell are stained green by a fluorescent dye able to permeate intact membranes. We only considered the changes in green fluorescence as a quantity that measures of total biomass produced on the surface, irrespective of the fact that this biomass consists of living and membrane compromised, dead cells. To that end, the fluorescence intensity of several fluorescence microscopy images was recorded at different time points, and that intensity was compared to the untreated silicon control surface (NC, =100% growth). For S. aureus (a), the data shows that substantially less biofilm is formed on the two polyzwitterions PSB and PZI, compared to the negative control. However, PSB, which is not antimicrobial, had 70% growth after 12 h compared to the negative control, while PZI had below 3%. On the polycationic SMAMP, on the other hand, the growth of S. aureus is dramatically increased compared to the negative control, which indicates that live bacteria can settle on the debris of the killed bacteria. The biofilm growth of E. coli (b) is significantly reduced on all test surfaces, 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.

[0162] FIG. 5: To test the biocompatibility of the PZI network, the Alamar Blue assay, (R. Hamid, Y. Rotshteyn, L. Rabadi, R. Parikh, P. Bullock, Toxicology in Vitro 2004, 18, 703-710) a standard cell toxicity assay, was used. In this assay, immortalized, non-carcinogenic human keratinocytes were seeded on the test surfaces, and cultivated. As living cells metabolize the Alamar Blue dye, the dye reduction compared to various controls is an indication of cell viability. FIG. 5 shows an Alamar Blue dye reduction (relative to initial dye concentration) by human keratinocytes grown for 24, 48 and 72 h, respectively, on PZI, SMAMP and PSB. The dye reduction by PZI and PSB was comparable to that of the growth control (neg). The dye reduction of the positive control corresponds to no cell growth. The figure shows the growth of keratinocytes on PZI within 72 h relative to a positive and negative control as defined in the Examples. The data shows that the cell growth is reduced to 49-55% of the value obtained for the negative control. Optical microscopy and fluorescence microscopy data (not shown) confirmed that the cells that grew on PZI were healthy and not membrane-compromised.

[0163] FIG. 6: shows FTIR spectra of the PZI network (a), the SMAMP network (b), and the PSB network (c.)

[0164] FIG. 7: illustrates the results from a 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 m; width: 33 m; spring constant: 18 Nm.sup.1; resonance frequency: 1400 kHz) and ScanAsyst Air cantilevers (length: 115 m; width: 25 m; spring constant: 0.4 Nm.sup.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 R from three images of an area of 55 m.sup.2 at different positions was taken.

[0165] FIG. 8: illustrates the results of protein adsorption on the PZI 6 and the two reference surfaces. The experiment was conducted as described. Right column: kinetics curve (reflectivity at constant angle vs. time). The first arrow marks the time point when protein (here: fibrinogen) was injected; the second arrow indicates the time point of buffer injection. Left column: reflectivity curves (reflectivity vs. angle) of the dry samples before (grey) and after (black dashed) protein adhesion. The data indicates that protein adhered strongly to the SMAMP, while both PSB and PZI 6 had quantitative protein repellency (within the accuracy limit of the method).

[0166] FIG. 9: shows representative potential titration curves of the PZI, SMAMP and PSB networks for PZI-networks, SMAMP-networks and PSB-networks

[0167] FIG. 10: shows the results from SPR Kinetics Experiments. For PZI-networks and PSB-networks as formed herein.

[0168] FIG. 11: demonstrates the effectiveness of PZI-networks against biofilm formation. As can be seen in FIG. 11, top, PZI significantly inhibited biofilm formation in the presence of S. aureus (see FIG. 11 Biofilm formation of S. aureus on PZI, SMAMP (=Butyl) and PSB (compared to the negative control (=growth control) after 12, 24, 48 and 72 h. The images are overlay of the green fluorescence and the red fluorescence. Biofilm formation was significantly inhibited in the presence of E. coli on PZI. Shown are also as comparative polymers SMAMP (=Butyl) and PSB, compared to negative control (compared to the negative control (=growth control) after 12, 24, 48 and 72 h. The images are an overlay of the green fluorescence and the red fluorescence.

[0169] FIG. 12: shows optical micrographs (phase contrast) of human keratinocytes grown on an uncoated glass slide (control, growth control), PSB and PZI after 24 h (A to C), 48 h (A to C) and 72 h (A to C). The cell density and the cell morphology on PSB and PZI is comparable to that on the growth control.

[0170] FIG. 13: shows the 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.

EXAMPLES

[0171] The examples shown in the following are merely illustrative and shall describe the present invention in a further way. These examples shall not be construed to limit the present invention thereto.

[0172] The herein presented data shows that the inventive antimicrobial and antifouling polymers and accordingly obtained polyzwitterion (PZI) coated surfaces (networks and monolayers) are strongly antimicrobial against both S. aureus and E. coli (as representatives of Gram-positive and Gram-negative bacteria, respectively). Even compared to the strongly antimicrobial SMAMP surface, PZI killed S. aureus more quantitatively, and the PZI biofilm mass was also significantly less on PZI than on SMAMP, because the debris of the dead bacteria cannot adhere as firmly to the PZI surface as it can stick to the SMAMP surface. Likewise, the data shows that PZI was strongly antimicrobial, protein-repellent and strongly reduced biofilm formation, while PSB was only protein-repellent and reduced biofilm formation less than the PZI. Hence, a superior antimicrobial and simultaneous antifouling property could be detected for the PZIs described herein. The instant invention thus presents the first polyzwitterions that are simultaneously antimicrobial and protein-repellent, and therefore significantly reduce bacterial biofilm formation. These represent highly promising material for medical applications.

Experimental Data

Example 1

General:

[0173] All chemicals were obtained as reagent grade from Sigma-Aldrich or Carl Roth and used as received. Dichloromethane was distilled from CaH.sub.2 under nitrogen. Solvents for gel-permeation chromatography (GPC) were HPLC quality and obtained from Carl Roth. Gel permeation chromatography (chloroform or THF, calibrated with PMMA standards) was measured on a PSS SDV or PSS GRAM column (PSS, Mainz, Germany). NMR spectra were recorded on a Bruker 250 MHz spectrometer (Bruker, Madison, Wis., USA). Electron ionization mass spectra were measured on a Thermo TSQ 700 spectrometer (Thermo Scientific, ionization energy 70 eV, source temperature 150 C.). Optical and fluorescence microscopy images were taken on a Nikon Eclipse Ti-S inverted microscope (Nikon GmbH, Dsseldorf, Germany).

Synthesis:

Synthesis of Crosslinking Agents:

[0174] The crosslinking agent 3EBP-silane was synthesized as described in the literature (see M. Gianneli, R. F. Roskamp, U. Jonas, B. Loppinet, G. Fytas, W. Knoll, Soft Matter 2008, 4, 1443). The crosslinking agent LS-BP was synthesized using the following procedure (Scheme 1):

##STR00008##

[0175] A solution of 4-hydroxybenzophenone (2.0 g, 10 mmol), lipoic acid (2.3 g, 11 mmol, 1.1 aq.) and DMAP (1.3 g, 11 mmol, 1.1 aq.) in anhydrous dichloromethane (DCM) was cooled by using an ice bath. Dicyclohexyl carbodiimide (DCC) (2.3 g, 11 mmol, 1.1 aq.) was dissolved in 10 ml anhydrous DCM and added within one hour. After stirring for 24 h at room temperature, the resulting urea byproduct was removed by filtration over a short silica gel column. The solvent was evaporated, and the crude product was purified by column chromatography (silica gel, ethyl acetate/n-hexane 1:3). The product 11 (2.4 g, 6.2 mmol, 62%) was obtained as a yellow solid.

##STR00009##

[0176] .sup.1H-NMR (250 MHz, CDCl.sub.3): 1.60 (m, 2H, 4-CH.sub.3), 1.72-1.85 (m, 4H, 3-CH.sub.2 & 5-CH.sub.2), 1.93 (dddd, 1H, 7-CH eq.), 2.48 (dddd, 1H, 7-CH ax.), 2.62 (t, 2H, 2-CH.sub.2), 3.14 (m, 2H, 8-CH.sub.2), 3.60 (ddt, 1H, 6-CH), 7.19-7.24 (m, 2H, 3-CH & 5-CH), 7.46-7.51 (m, 2H, 3-CH & 5-CH), 7.56-7.62 (m, 1H, 4-CH), 7.78-7.82 (m, 2H, 2-CH & 6-CH), 7.83-7.88 (m, 2H, 2-CH & 6-CH). .sup.13C-NMR (62.9 MHz, CDCl.sub.3): 25.0 (2-CH.sub.2), 29.1 (4-CH.sub.2), 34.6 (2-CH.sub.2), 35.0 (5CH.sub.2), 38.9 (8-CH.sub.2), 40.7 (7-CH.sub.2), 56.7 (6-CH), 121.9 (3-CH), 128.8 (2-CH), 130.4 (3-CH), 132.1 (2-CH), 132.9 (4-CH), 135.4 (1-C), 137.9 (f-C), 154.3 (4-CO), 171.9 (1-CO), 195.9 (OCPh.sub.2).

[0177] The compounds were used for surface attachment of the herein described PZI network structures and polymers.

Synthesis of Zwitterion Precursor Monomer:

[0178] 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), which was dissolved in CH.sub.2Cl.sub.2. 1.1 eq of N-(tert-butoxycarbonyl)ethanolamine (5.32 g, 33 mmol) and 10 mol % 4-dimethylaminopyridine (DMAP) were added. After stirring over night, the solution was washed with 10% KHSO.sub.4 and water, and dried with Na.sub.2SO.sub.4. 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%.

##STR00010##

[0179] .sup.1H-NMR (250 MHz, CDCl.sub.3): 1.46 (s, 9H, 9-CH.sub.3), 2.87 (m, 2H, 3-CH & 3-CH), 3.38 (m, 2H, 6-CH.sub.2), 4.03-4.31 (m, 2H, 5-CH.sub.2), 5.13 (br s, 1H, NH), 5.26 & 5.34 (m, 2H, 2-CH & 2-CH), 6.47 (m, 2H, 1-CH & 1-CH), 6.81-7.02 (br s, 1H, OH).

[0180] .sup.13C-NMR (62.9 MHz, CDCl.sub.3): 28.35 (C9), 39.60 (C6), 46.78 & 47.35 (C3, C3), 64.44 (C5), 80.08 & 80.52 (C2, C2), 136.41 & 136.58 (C1, C1), 171.44 & 174.59 (C4, C4).

[0181] MS: m/z=328.14 (M.sup.+.), 283.09 (M-COOH), 272.07 (M-tBu), 226.03 (M-CO.sub.2tBu).

Polymerization of Zwitterion Precursor Monomer:

[0182] The polymerization of the zwitterion precursor monomer was performed under nitrogen using standard Schlenk techniques. The zwitterion monomer precursor (500 mg, 1.2 mmol) was dissolved in 5 mL THF. The Grubbs third generation catalyst (3.7 mg, 5 mol) was dissolved in 2 mL CH.sub.2Cl.sub.2 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 or 2 hours. 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. GPC was performed in THF (calibrated with PMMA standards).

##STR00011##

[0183] GPC: M.sub.n=73,000 g/mol, M.sub.w=iii 200 g mol.sup.1, PDI=1.5

[0184] .sup.1H-NMR 250 MHz, THF-d.sub.8): 1.41 (s, 9H, H9), 3.10 (br m, 2H, H3 & H3), 3.28 (br m, 2H, H6), 4.08 (br m, 2H, H5), 4.67 (m, 1H, H2 trans), 5.10 (br s, 1H, H2 cis), 5.59 (br s, 1H, NH), 5.88 (br m, 1H, H1 cis) and 6.08 (br m, 1H, H1 trans).

[0185] .sup.13C-NMR (75 MHz, THF-d.sub.8): .sup.13C-NMR (62.9 MHz, THF-d.sub.8): 28.93 (C9), 40.37 (C6), 53.83 & 54.40 (C3, C3), 64.98 (C5), 78.93 (BOCCCH.sub.3), 81.44 & 81.88 (C2, C2), 133.42 & 133.60 (C1, C1), 156.86 (NCO) 171.36 & 172.72 (C4, C4).

Polyzwitterion Precursor Polymer Deprotection:

[0186] 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, 5-10 vol % dry methanol were added to maintain solubility of the hydrolyzing polymer. The mixture was stirred for 18 hours at room temperature. The solvent was removed and the precipitate was re-dissolved in methanol. It was purified by precipitation into ice-cooled diethyl ether. Up to 10 vol % n-hexanes were added in case the polymer did not precipitate.

##STR00012##

[0187] .sup.1H-NMR (250 MHz, MeOH-d.sub.4): 3.35 (br s, 2H, H3 & H3+solvent), 3.72 (br s, 2H, H6), 4.40 (br s, 2H, H5), 4.74 (m, 1H, H2 trans+solvent), 5.16 (br s, 1H, H2 cis), 5.73 (br s, 1H, H1 cis) and 5.97 (br s, 1H, H1 trans).

[0188] .sup.13C-NMR (62.9 MHz, MeOH-d.sub.4): 40.10 (C6), 53.42 & 53.99 (C3, C3), 62.76 (C5), 78.63 & 82.43 (C2, C2), 132.66 & 133.65 (C1, C1), 173.06 & 172.28 (C4, C4).

Synthesis of Butyl SMAMP Monomer:

[0189] The Butyl monomer was synthesized and characterized as previously published (see K. Lienkamp, A. E. Madkour, A. Musante, C. F. Nelson, K. Nuesslein, N. Tew Gregory, J Am Chem Soc 2008, 130, 9836).

Polymerization of Butyl SMAMP Monomer:

[0190] 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 CH.sub.2Cl.sub.2. Grubbs third generation catalyst (0.72 mg, 1.1 mol) was dissolved in 1 mL CH.sub.2Cl.sub.2 in a second flask and added in one shot to the vigorously stirring monomer solution at room temperature under N.sub.2. After 30 min, excess ethylvinyl ether (1 mL, 750 mg, 10 mmol) was added. The mixture was stirred for 2 hours. The solvent was then evaporated under reduced pressure. The product was re-dissolved in a small amount of dichloromethane and added dropwise into ice-cooled n-hexane. The colorless precipitate was removed by filtration, and dried in dynamic vacuum. The NMR signals of the polymer matched those in the literature (see K. Lienkamp, A. E. Madkour, A. Musante, C. F. Nelson, K. Nsslein, G. N. Tew, Journal of the American Chemical Society 2008, 130, 9836).

[0191] GPC analysis (PSS SDV column, Chloroform, r.t., 1 mL min.sup.1): M.sub.n=235,900 g mol.sup.1, M.sub.w=260,100 g mol.sup.1, PDI=1.1.

Synthesis of PSB Monomer:

[0192] The PSB monomer was synthesized and characterized as previously published (see S. Colak, G. N. Tew, Langmuir 2012, 28, 666).

Polymerization of PSB Monomer:

[0193] The polymerization of the PSB monomer was performed under nitrogen using standard Schlenk techniques.

Synthesis of the Benzophenone-Containing Monomer for the Internal Crosslinker (N-(2-(4-oxobenzophenone)-ethyl)-3,6-tetrahydrophthalimide)

[0194] ##STR00013##

[0195] 4-Hydroxybenzophenone (19.88 g, 0.1 mol, 10 eq) and K.sub.2CO.sub.3 (13.82 g, 0.1 mol, 10 eq) were dissolved in 250 mL dry acetone and the reaction mixture was heated to the boiling temperature. Then N-(2-bromoethyl)-3,6-tetrahydrophthalimide (2.72 g, 0.01 mol, 1 eq) was slowly added. The reaction mixture was refluxed for 24 h. After cooling down, water was added and the solution was extracted with DCM. The combined organic phases were washed with 10% NaOH (3). The organic phase was dried and the solvent was evaporated under reduced pressure. After removal of the solvent, the residue was not solid. NMR was taken and the oil was recrystallized via dissolving the residue in small amounts of DCM and layering with hexane. The flask was stored in freezer until crystallization occurred. The product was filtered off, washed with cold hexanes, and dried.

##STR00014##

[0196] .sup.1H NMR (250 MHz, acetone): 7.44-7.89 (m, 7H), 7.04 (d, J=8.85 Hz, 2H), 6.58 (s, 2H), 5.16 (s, 2H), 4.25 (t, J=5.84 Hz, 2H), 3.88 (t, J=5.80 Hz, 2H), 2.96 (s, 2H)

[0197] .sup.13C NMR (62.86 MHz, acetone): 195.3 (1C), 177.1 (1C), 163.1 (1C), 139.3 (1C), 137.5 (2C), 133.1 (2C), 132.8 (1C), 131.4 (1C), 130.3 (2C), 129.2 (2C), 115.2 (2C), 81.9 (1C), 65.2 (1C), 48.4 (2C), 38.4 (1C)

Synthesis of the Internal Crosslinker by Copolymerization of (N-(2-(4-oxobenzophenone)-ethyl)-3,6-tetrahydrophthalimide) with the Inventive Monomers

[0198] A stock solution of Grubbs' catalyst was prepared in a flame dried flask. The benzophenone monomer (295 mg, 0.9 mmol, 0.9 eq) and the zwitterion monomer (39 mg, 0.1 mmol, 0.1 eq) were added to a flame dried flask and dissolved in 10 mL anhydrous THF under nitrogen atmosphere. After 30 min of stirring, 2 mL of the stock solution (c=2.4 mg/mL) was added in one shot. The reaction mixture was stirred for another 60 minutes. Then 1 mL of ethyl vinyl ether was added to terminate the reaction. After another 30 minutes the polymer was precipitated in diethylether. The polymer was dried under high vacuum overnight. NMR showed traces of diethyl ether. Therefore the product was further dried.

##STR00015##

[0199] .sup.1H NMR (250 MHz, THF): 7.31-7.87 (m, 7H, benzophenone), 6.93-7.10 (m, 2H, benzophenone), 50.85-6.20 (trans) and 5.59 (cis) (br, 2H total), 50.10 (cis) and 4.67 (trans) (br, 2H total), 4.18-4.40 (m, 2H, bBenzophenone), 4.07 (br, 2H), 3.79-3.97 (m, 2H, benzophenone), 3.28 (br, 2H), 3.11 (br, 2H), 1.41 (s, 9H)

Derivatization for GPC Measurement:

[0200] 20 mg of the internal crosslinker polymer were dissolved in 1 mL THF (anh.) and 1 mL MeOH (anh.). Then 2 mL of trimethylsilyl diazomethane (2M in diethylether) were added. After 30 minutes the solution was evaporated and the remaining polymer was used for GPC analysis.

[0201] GPC analysis (PSS SDV column, Chloroform, r.t., 1 mL min.sup.1): M.sub.n=38,000 g mol.sup.1, M.sub.w=46,000 g mol.sup.1, PDI=1.2.

Functionalization of Silicon Wafer and Gold Substrate with Crosslinking Agents:

[0202] Silicon wafer: A solution of 3EBP-silane (20 mg mL.sup.1 in toluene) was spin coated on a 52525 m 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.

Gold:

[0203] For SPR measurements, the LaSFN9 glass slides coated with a 1 nm chromium layer 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.

[0204] Wound dressing: Commercially available polyurethane-based wound dressing foam (Suprasorb P, Lohmann and Rauscher, Rengsdorf, Germany, 5 mm thickness) was cut into 22 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 (see also FIG. 2).

Immobilization of the Inventive PZI Polymer (and the Reference Polymers) as Surface-Attached Polymer Network on Silicon Wafers, Gold and Glass Substrates that have been Functionalized with a Molecule for Surface Attachment Containing the Photo-Crosslinking Unit Benzophenone, Using the External Crosslinker Pentaerythritol-Tetrakis-(3-Mercaptopropionate) Carrying Thiols as Photo-Crosslinking Units:

Procedure Using the Protected PZI:

[0205] A stock solution (Solution A) was prepared by dissolving pentaerythritol-tetrakis-(3-mercaptopropionate) mL, 1.3 g, 2.6 mmol) in THF (50 mL). The poly(zwitterion) precursor (10 mg, 0.027 mmol) was dissolved in Solution A (0.25 mL). Chloroform (0.4 mL for silicon coating or 0.8 mL for gold and 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 or LS-BP treated gold substrate at 3000 rpm for 30 sec. The film was crosslinked at 254 nm for 30 min in a BIO-LINK Box (Vilber Lourmat GmbH). It was then washed with THF to remove unattached polymer chains and dried overnight under N.sub.2-flow. This yielded the precursor poly(zwitterion) network. To remove the Boc protective groups, function, the film was immersed in HCl (4 M in dioxane) for 12 hours and washed twice with ethanol. It was then dried over night under N.sub.2-flow to yield the PZI network.

Procedure for Direct Coating with Deprotected PZI:

[0206] A stock solution of crosslinker was prepared by dissolving pentaerythritol-tetrakis-(3-mercaptopropionate) (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 crosslinker solution in ethyl acetate was added and the 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 crosslinked at 254 nm for 30 min in a BIO-LINK Box (Vilber Lourmat GmbH). It was then washed with methanol to remove unattached polymer chains and dried under N.sub.2-flow.

Butyl SMAMP Network:

[0207] A stock solution (Solution B) was prepared by dissolving pentaerythritol-tetrakis-(3-mercaptopropionate) (1 mL, 1.3 g, 2.6 mmol) in CH.sub.2CL.sub.2 (50 mL). The precursor Butyl SMAMP polymer (10 mg, obtained as described in P. Zou, D. Laird, E. K. Riga, Z. Deng, H.-R. Perez-Hernandez, D. L. Guevara-Solarte, T. Steinberg, A. Al-Ahmad, K. Lienkamp, Journal of Materials Chemistry B 2015, 3, 6224-6238) was dissolved in Solution B (0.25 mL). Toluene (0.3 mL) was added as co-solvent, and the mixture was stirred for 60 s. The remaining steps were exactly the same as described above for the poly(zwitterion) network.

PSB Network:

[0208] 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, obtained as described in S. Colak, G. N. Tew, Langmuir 2012, 28, 666-675) 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 crosslinked at 254 nm for 30 min in a BIO-LINK Box (Vilber Lourmat GmbH). It was then washed with TFE to remove unattached polymer chains and dried under N.sub.2-flow.

Immobilization of the Inventive PZI Polymer as Surface-Attached Polymer Network on Silicon Wafers, Gold and Glass Substrates that have been Functionalized with a Molecule for Surface Attachment Containing the Photo-Crosslinking Unit Benzophenone, Using the Internal Crosslinker Containing (N-(2-(4-Oxobenzophenone)-Ethyl)-3,6-Tetrahydrophthalimide)) Carrying Benzophenone as Photo-Crosslinking Units:

[0209] 10 mg of the internal crosslinker containing N-(2-(4-oxobenzophenone)-ethyl)-3,6-tetrahydrophthalimide were dissolved in 1 mL of THF. The solution was spin coated on a 3-EBP treated silicon wafer at 3000 rpm for 30 s. The film was crosslinked at 254 nm with 3 J/cm.sup.2. in a BIO-LINK Box (Vilber Lourmat GmbH). The film thickness d was determined by ellipsometry.

[0210] d=585 nm

Polymer Network Characterization:

Ellipsometry:

[0211] The thickness of the dry polymer layers on silicon wafers was measured with the auto-nulling imaging ellipsometer Nanofilm EP.sup.3 (Nanofilm Technologie GmbH, Gttingen, 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.

[0212] PZI network: 861 nm

[0213] Butyl SMAMP network: 152 nm1 nm

[0214] PSB network: 71 nm3 nm

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy (ATR-FTIR):

[0215] Double side polished silicon wafers were used as substrates for the FTIR experiments. The polymer layer was immobilized on one side of a double side polished silicon wafer. The spectra were recorded from 4000 to 400 cm.sup.1 with a Bio-Rad Excalibur spectrometer (Bio-Rad, Mnchen, Germany), using a spectrum of the blank double side polished silicon wafer as background. Spectra of the different test samples are shown in FIGS. 6a), b) and c).

Atomic Force Microscopy (AFM):

[0216] The topography of the surfaces was imaged with a Dimension FastScan and Icon from Bruker. Commercial FastScan-A cantilevers (length: 27 m; width: 33 m; spring constant: 18 Nm.sup.1; resonance frequency: 1400 kHz) and ScanAsyst Air cantilevers (length: 115 m; width: 25 m; spring constant: 0.4 Nm.sup.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 R from three images of an area of 55 m.sup.2 at different positions was taken. The images are shown in FIG. 7.

Contact Angle:

[0217] 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.

TABLE-US-00001 Static/ Advancing/ Receding/ PZI network 21 2 37 1 14 1 Butyl SMAMP network 70 3 68 3 17 2 PSB network 37 2 56 2 22 2

Zeta Potential Measurements:

[0218] 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, 20101 mm lp, Ch.Nr. 13112704) and put into the measuring cell.

[0219] Before each measurement the electrolyte hoses were rinsed with ultrapure water until a conductivity of <0.06 mS m.sup.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 m while the system was rinsed for 180 sec. at 300 mbar.

[0220] 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 180 sec. 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.

SPR (Surface Plasmon Resonance) Measurements and Sample Preparation.

[0221] SPR measurements were performed on a RT2005 RES-TEC device in Kretschmann configuration from Res-Tec, Framersheim, Germany. Excitation was done with a HeNe-Laser with =632.8 nm. SPR substrates were homemade (LaSFN9 glass from Hellma GmbH, Mllheim, 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). (see FIG. 8).

SPR Angular Scans.

[0222] To study the build-up of the material, a full reflectivity curve was measured after each fabrication step. Before and after the adsorption experiments in the kinetics mode, full angular scans of the dry substrates were also measured.

SPR Kinetics Experiments.

[0223] Protein adsorption was studied in the kinetics mode. To set up the experiment, an angular scan of the substrate under HEPES flow was performed to detect the minimum. The protein adsorption experiments in the kinetic mode were then carried out at .sub.exp=.sub.min1 and a flow rate of 50 l h.sup.1 of the fibrinogen solution (1 mg ml.sup.1). To determine the thickness of adsorbed fibrinogen after the kinetics experiment, the surfaces were rinsed with MilliQ water for 15 min to remove residual salt and dried under nitrogen flow. Afterwards a reflectivity curve was measured again. The thickness of each protein layer was calculated by simulations based on the Fresnel equations, which were performed with the software Winspall (see FIG. 10).

PZI Network:

[0224] The following permittivities and were used: LaSFN9 (=3.4036; =0); Cr (=6.3; =20); Au (=12.3; =1.16) PZI (=2.43; =0), LS-BP, fibrinogen (=2.25; =0), nitrogen (=1; =0).

Poly(Sulfobetaine) (PSB) Network:

[0225] The following permittivities and were used: LaSFN9 (=3.4036; =0); Cr (=6.3; =20); Au (=12; =1.3) PSB (=2.25; =o), LS-BP, fibrinogen (=2.25; =0), nitrogen (=1; =0).

Determination of the Swelling Ratio of Surface Attached Polymer Networks

[0226] For the swelling experiments, thicker samples (about 250 to 2000 nm) 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. Each curve was simulated with Fresnel calculations and the two unknown parameters, the sample thickness d and the real part of the permittivity () 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:

[00001]$Q=\frac{d_{\mathrm{solvent}}}{d_{\mathrm{dry}}}$

[0227] The SPR reflectivity curves (grey) are shown together with the simulation curves (black dashed) for each polymer layer. The respective layer thickness and real permittivity are listed below the curves.

[0228] The physical characterization of the exemplary surface-attached PZI network 6 is displayed in table 1. The dry layer thickness and the swellability ratio (=waterswollen layer thickness/dry layer thickness) were obtained by ellipsometry measurements; the fibrinogen adhesion was quantified using surface plasmon resonance spectroscopy; the isoelectric point was determined using the SurPass surface analyzer (Anton Paar, Austria). The data shows that the PZI-coated surface 6 was strongly protein-repellent.

TABLE-US-00002 TABLE 1 Diy Layer Swellability Fibrinogen Iso-electric Thickness/nm Ratio/H.sub.2O adhesion/ng mm.sup.2 point PZI 86 1 1.9 0 0 6.6 0.1

Polymer Network Characterization:

Antimicrobial Activity Assay.

[0229] The experiments 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 (see a) A. Al-Ahmad, P. Zou, D. L. Guevara Solarte, E. Hellwig, T. Steinberg, K. Lienkamp, PLoS One 2014, 9, e111357/1; b) J. Haldar, A. K. Weight, A. M. Klibanov, Nature Protocols 2007, 2, 2412). S. aureus (ATCC29523) and E. coli (ATCC25922) were cultured overnight in triptic soy broth and diluted 1:10. Optical density was checked 3-4 hours later and the bacterial culture (1.5 ml of S. aureus and 150 L of E. coli) was mixed in a chromatography sprayer bottle with 100 ml of sterile NaCl 0.9% solution and continuously stirred (see a) A. Al-Ahmad, P. Zou, D. L. Guevara Solarte, E. Hellwig, T. Steinberg, K. Lienkamp, PLoS One 2014, 9, e111357/1). The test samples (5 of each material), including positive and negative controls, were fixed at the center of sterile Petri dishes each and placed at a distance of 15 cm to the spray nozzle. Then the bacterial suspension was sprayed onto the samples using compressed air from a 50 mL syringe (see P. Zou, D. Laird, E. K. Riga, Z. Deng, F. Dorner, H.-R. Perez-Hernandez, D. L. Guevara-Solarte, T. Steinberg, A. Al-Ahmad, K. Lienkamp, Journal of Materials Chemistry B 2015, 3, 6224). Afterwards, the petri dishes were immediately covered and incubated for 2 h in a humid chamber at 37 C. under aerobic conditions and 5% CO.sub.2. 50 L of sterile 0.9% NaCl solution was added onto the samples and left for 2 min. To ensure removal of bacteria from the surface the solution was pumped back and re-pipetted twice and spread over Columbia blood agar plates. These were incubated overnight at 37 C. without agitation. CFUs were counted with the software Quantitiy One Each experiment was tested at least twice.

Biofilm Formation Studies

[0230] The test samples were silicon wafer pieces coated with the different polymer networks cut to a size of 55 mm. The growth control was an uncoated silicon wafer piece cut to a size of 55 mm. The samples and control pieces were placed in the wells of a sterile 24-wellplate using sterilized tweezers. 1000 L of bacterial overnight culture (106 bacteria cm-3) in tryptic soy broth (TSB) medium was added to each well. The bacteria tested in each experiment were: S. aureus ATCC29523, E. coli (ATCC25922).

[0231] All the samples sets were incubated (at 37 C. with 5% CO.sub.2) without agitation for 2 h, then 500 L 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 and the growth control were placed in a new microplate and all of them were washed 3 times with NaCl (0.9%) in order to remove the non-adherent microorganisms.

[0232] Life/dead staining (Live/Dead BacLight bacterial viability kit, Molecular Probes, Eugene, Oreg., USA) was used according to the instructions of the manufacturer in each well, and the samples were stored for 10 min in a dark chamber; after that every sample was placed face down in an Ibidi -Slide 8 well chamber, and imaged using the Fluorescent microscope (Zeiss Axio Observer Z1) with a 63 objective. The excitation/emission maxima for these dyes were 500 nm for SYTO 9 (green-fluorescent) stain and 617 nm for propidium iodide (red-fluorescent) stain.

[0233] Results of the biofilm formation are shown in FIG. 11 (top and bottom). As can be seen in FIG. 11, top, PZI significantly inhibited biofilm formation in the presence of S. aureus (see FIG. 11 biofilm formation of S. aureus on PZI, SMAMP (=Butyl) and PSB (compared to the negative control (=growth control) after 12, 24, 48 and 72 h. The images are overlay of the green fluorescence and the red fluorescence. Moreover, as can be seen in FIG. 11 bottom, biofilm formation was significantly inhibited in the presence of E. coli on PZI, SMAMP (=Butyl) and PSB (compared to the negative control (=growth control) after 12, 24, 48 and 72 h. The images are an overlay of the green fluorescence and the red fluorescence.

Alamar Blue Assay.

Sample Preparation:

[0234] Alamar Blue 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 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% isopropyl alcohol to emulate the process used for the spin-coated 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 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:

[0235] Immortalised HPV-16 gingival mucosal keratinocyte (GM-K) cells [7] were cultivated in Keratinocyte Growth Medium (KGM) (Promocell, Heidelberg, Germany) with accompanying supplements prepared at concentrations supplied by the manufacturer: bovine pituitary extract0.004 ml/ml; epidermal growth factor (EGF)0.125 ng mL.sup.1; insulin5 g mL.sup.1; hydrocortisone0.33 g mL.sup.1; epinephrine0.39 g mL.sup.1; transferrin10 g mL.sup.1; CaCl.sub.20.06 mM; in addition to the antibiotic kanamycin at 100 g mL.sup.1. Cells were trypsinized 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 1.510.sup.5 cells mL.sup.1 in supplement/antibiotic free medium. Thereafter, the 12 well plates containing the cells were incubated at 37/5% CO.sub.2 for 5 hours allowing cells to settle and begin adhesion. At this time 500 L of medium above the cells was carefully aspirated and replaced by 500 l medium containing double normal supplement concentration yielding a normal supplement concentration medium. Cells on test surfaces and controls were cultivated for a further 18 hours (total 24 hours), 42 h (total 48 h) and 66 h (total 72 h). At each time point positive control samples were generated by aspirating 500 L medium from 3 wells and adding 500 L 60% iso-propyl alcohol to give a 30% iso-propyl alcohol solution. A negative control was generated by removing the old medium and replacing it with 1 mL fresh medium. All samples and controls were cultivated for a further 30 minutes, after which 110 L 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 returned to the incubation chamber for 2 hours, after which time all medium containing Alamar Blue was aspirated and collected into 1.5 ml Eppendorf tubes. Tubes were centrifuged at 1,000 g for 5 minutes to exclude cells, then the fluorescence intensity of the supernatant was measured (excitation at 540 nm and measurement at 590 nm) on a Tecan, Infinte 200 plate reader and data 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.

[0236] Results are shown in FIGS. 5, 12 and 13. FIG. 5 shows an Alamar Blue dye reduction (relative to initial dye concentration) by human keratinocytes grown for 24, 48 and 72 h, respectively, on PZI, SMAMP and PSB. The dye reduction by PZI and PSB was comparable to that of the growth control (neg). The dye reduction of the positive control corresponds to no cell growth. FIG. 12 shows optical micrographs (phase contrast) of human keratinocytes grown on an uncoated glass slide (control, growth control), PSB and PZI after 24 h (A to C), 48 h (A to C) and 72 h (A to C). The cell density and the cell morphology on PSB and PZI is comparable to that on the growth control. Finally, FIG. 13 shows the 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.

Example 2

Synthesis of Itaconic Acid 4-propyl-amide

[0237] Itaconic anhydride (5.0 g, 44.6 mmol) was dissolved in dichloromethane (DCM) (20 mL) and H.sub.2SO.sub.4 (conc., 0.1 mL) was added. The solution was ice cooled for 15 min. Then the n-propylamine (4.0 mL, 2.9 g, 49.1 mmol, 1.1 eq) in DCM (10 mL) was added dropwise over 30 min. After another 10 min the ice bath was removed and the solution was stirred overnight at room temperature. Subsequently the precipitate (pure product) was removed by filtration and dried at dynamic vacuum overnight. The product was obtained as colorless solid. The structure of the obtained monomer and the proton numbering for .sup.1H-NMR assignment are shown below.

##STR00016##

[0238] Yield: 3.9 g; 22.8 mmol; 50%

[0239] MS (ESI, 4-5 kV): m/z={[M+Na]}=194.08

[0240] .sup.1H NMR (250 MHz, Acetone-d6): =11.46 (br. s, OH), 7.24 (br. s., NH), 6.22 (s, 1-H), 5.73 (s, 1-H), 3.24 (s, 3-CH.sub.2), 3.17 (td, J=6.50 Hz, 5-CH.sub.2), 1.51 (tq, J=7.30 Hz, 6-CH.sub.2), 0.90 (t, J=7.42 Hz, 7-CH.sub.3).

[0241] .sup.13C NMR (63 MHz, Acetone-d6): =170.22 (s, 4-CO), 167.57 (s, 2-CO), 136.42 (s, 2-C), 127.38 (s, 1-C), 41.18 (s, 5-C), 39.40 (s, 3-C), 22.92 (s, 6-C), 11.13 (s, 7-C).

Example 3

Synthesis of Itaconic Acid 1-(N-Boc-aminoethyl)-4-propyldiamide

[0242] The reaction was performed under nitrogen atmosphere. N-Boc-ethylenediamine (2.2 g, 14.0 mmol, 1.2 eq) and DMAP g, 1.2 mmol, 0.1 eq) were added to a solution of the itaconic acid 4-propylamide (2.0 g, 11.6 mmol) in DCM (30 mL). The solution was ice cooled for 10 min, then 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (2.2 g, 14.0 mmol, 1.2 eq) was added to the reaction solution in one portion. The solution was stirred overnight at room temperature. Then the reaction mixture was washed with HCl (1M, 250 mL), aqueous NaHCO.sub.3 (saturated, 250 mL), aqueous NaCl (saturated, 150 mL) and water (150 mL).

[0243] It was then dried over Na.sub.2SO.sub.4 and the solvent was evaporated at the rotary evaporator. The product was dried at dynamic vacuum overnight to yield a colorless solid.

##STR00017##

[0244] Yield: 2.21 g, 7.1 mmol, 61%

[0245] MS (ESI, 4-5 kV): m/z={[M+Na].sup.+}=336.19

[0246] .sup.1H NMR (250 MHz, Acetone-d.sub.6) =7.89 (br. s., NH), 7.35 (br. s., NH), 6.14 (br. s., NH), 5.84 (s, 1-H), 5.46 (s, 1-H), 3.36 (td, J=6.10 Hz, 4-CH.sub.2), 3.26 (t, J=6.10 Hz, 5-CH.sub.2), 3.20 (s, 3-CH.sub.2), 3.14 (td, J=7.00 Hz, 5-CH.sub.2), 1.50 (tq, J=7.20 Hz, 6-CH.sub.2), 1.42 (s, 9-(CH.sub.3).sub.3), 0.90 (t, J=7.42 Hz, 7-CH.sub.3).

[0247] .sup.13C-NMR (63 MHz, Acetone-d.sub.6): =170.18 (s, 3-CO)**, 168.39 (s, 4-CO)**, 156.61 (s, 7-CO), 134.08 (s, 2-C), 129.32 (s, 1-CH.sub.2), 78.25 (s, 9-C(CH.sub.3).sub.3), 51.93 (s, 5-CH.sub.2), 48.62 (s, 4-CH.sub.2), 40.40 (s, 5-CH.sub.2), 34.06 (s, 3-CH.sub.2), 28.13 (s, 9-C(CH.sub.3).sub.3), 22.98 (s, 6-CH.sub.2), 11.15 (s, 7-CH.sub.3).

Example 4

Copolymers: Synthesis of Poly[(itaconic acid 1-(N-boc-aminoethyl).SUB.4.-propyldiamide)-co-(N,N-dimethyl acrylamide)]

[0248] The copolymerization of 1-(2-N-Boc-ethyl)-4-propyldiitaconamide with N,N-Dimethylacrylamide (DMAA) at the ratio 50 mol % of each comonomer was performed under nitrogen atmosphere. The diitaconamide (to g, 3.2 mmol, 0.5 eq), DMAA (0.3 mL, 0.3 g, 3.2 mmol, 0.5 eq) and AIBN (5.3 mg, 0.003 mmol, 0.1 mol %) were dissolved in N,N-Dimethylformamide (DMF) (1.6 mL). The reaction mixture was subject to three freeze-pump-thaw cycles and then stirred at 70 C. for 22 h. The mixture was then cooled and the reaction was quenched by stirring the open flask under ambient atmosphere. Subsequently the solvent was removed under reduced pressure, the product was diluted in DCM (10 mL) and then added dropwise into n-hexane (100 mL) while stirring vigorously. The polymer precipitated. It was removed by filtration The precipitation was performed three times. The product was obtained as slightly yellow solid and dried at dynamic vacuum overnight. The yield and the GPD data are given below. The structure of the obtained polymers obtained and the proton numbering for .sup.1H-NMR assignment are shown below.

##STR00018##

Poly[(itaconic acid 1-(N-Boc-aminoethyl).SUB.4.-propyl diamide)-co-DMAA]

[0249]

TABLE-US-00003 Ester to DMAA Yield/ M.sub.n/ M.sub.w/ Copolymer ratio mg Yield/% g mol.sup.1 g mol.sup.1 PDI Propylamide- 0.5/0.5 495 70 7,000 34,500 3.0 co-DMAA .sup.1H NMR Propylamide-co-DMAA (250 MHz, CDCl.sub.3): 3.56-2.71 (m, 12H), 2.06 (br.s, 2H) 1.67-1.26 (m, 11H), 0.94 (br.s, 3H).

Example 5

Synthesis of Itaconic Acid 4-(N-Boc-aminoethyl)-amide

[0250] Itaconic anhydride (1.0 g, 8.9 mmol) was dissolved in DCM (30 mL) and ice cooled for 15 min. Then the N-Bocethylenediamine (1.6 g, 9.8 mmol, 1.1 eq) in DCM (10 mL) was added dropwise over 20 min. After another 10 min the ice bath was removed and the solution stirred for 1 h at room temperature. Subsequently the precipitate (pure product) was removed by filtration and dried at dynamic vacuum overnight. The product was obtained as colorless solid. The structure of the obtained monomer and the proton numbering for .sup.1H-NMR assignment are shown below.

##STR00019##

[0251] Yield: 2.8 g; 10.3 mmol; 58%

[0252] MS (ESI, 4-5 kV): m/z={[M+Na]}=295.13

[0253] .sup.1H NMR (250 MHz, dmso-d.sub.6): =12.46 (br. s, OH), 7.87 (br. s., NH), 6.77 (br. s., NH), 6.11 (s, 1H), 5.66 (s, 1H), 3.08 (s, 3-H), 3.05-2.95 (m, 5-H, 6-H), 1.39 (s, 7-(CH.sub.3)3, 9H).

[0254] .sup.13C NMR (63 MHz, methanol-d.sub.4): =172.41 (s, CO), 168.61 (s, CO), 157.53 (s, CO), 135.66 (s, CC), 128.14 (s, CC), 79.16 (s, C(CH.sub.3).sub.3), 39.82 (s, CH.sub.2), 39.67 (s, CH.sub.2), 39.18 (s, CH.sub.2), 29.75 (s, C(CH.sub.3).sub.3).