WO2017035566A1 - Plasma polymerised oxazoline coatings and uses thereof - Google Patents

Abstract

A plasma polymerised polyoxazoline polymer is disclosed. Also disclosed is a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof, a process for preparing a plasma polymerised polyoxazoline polymer film on a surface of a substrate, a substrate comprising an antibiofouling surface, a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof and one or more ligands and/or biomolecules bonded to the polyoxazoline polymer film, and a process for immobilising a biomolecule on a surface of a substrate.

Description

PLASMA POLYMERISED OXAZOLINE COATINGS AND USES THEREOF

PRIORITY DOCUMENT

[0001 ] The present application claims priority from Australian Provisional Patent Application No. 2015903578 titled "PLASMA POLYMERISED OXAZOLINE COATINGS AND USES THEREOF" and filed on 2 September 2015, the content of which is hereby incorporated by reference in its entirety.

FIELD

[0002] The present disclosure relates to polyoxazoline coatings and uses of substrates coated with polyoxazolines.

BACKGROUND

[0003 ] The synthesis of polyoxazolines (POx), via ring opening solution polymerisation, was first reported in 1966 by four independent research groups [1 -4]. Early research on these compounds highlighted their potential for use as stabilisers, adhesives, surfactants and dispersants. However, industrial applications involving this family of compounds did not develop, most likely due to the complex, lengthy (up to a month reaction time at ambient temperature) and costly synthesis methods which made them unattractive for batch production compared to competing polymers.

[0004] Over the last decade polyoxazoline polymers have had a renaissance because of the discovery of some interesting properties such as low biofouling [5 ] [6] and good biocompatibility [7] which makes them ideal candidates for application in areas such as delivery of therapeutics, biomaterials and implant technologies. With this renewed interest, many research groups have attempted to overcome some of the synthesis drawbacks. The major improvement was made by Hoogenboom and co-workers who significantly decreased the synthesis time by introducing microwave assisted polymerisation in 2005. Despite these advances, bulk polyoxazoline synthesis in solution remains a long, multistep organic chemistry process conducted in organic solvents. The synthesis also generates waste products and impurities [8 |.

[0005] There is a need for processes for producing polyoxazoline polymers that overcome one or more of the difficulties associated with known production processes.

SUMMARY

10006] According to a first aspect, there is provided a plasma polymerised polyoxazoline polymer. [0007] According to a second aspect, there is provided a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof.

[0008] According to a third aspect, there is provided a process for preparing a plasma polymerised polyoxazoline polymer film on a surface of a substrate, the process comprising exposing the surface of the substrate to a plasma comprising an oxazoline monomer vapour under conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazol ine polymer on the surface of the substrate.

[0009] Advantageously, the plasma polymerised polyoxazoline polymer prevents, reduces or minimises biofouling of surfaces onto which it is coated. Therefore, according to a fourth aspect there is provided a substrate comprising an antibiofouling surface, wherein the anti biofouling surface comprises a plasma polymerised polyoxazoline polymer.

[0010] Ligands and biological molecules can also be immobilised on substrates comprising a plasma polymerised polyoxazoline polymer film on a surface thereof. Therefore, according to a fifth aspect, there is provided a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof and one or more ligands and/or biomolecules bonded to the polyoxazoline polymer film.

[001 1 ] According to a sixth aspect, there is provided a process for immobilising a biomolecule on a surface of a substrate, the process comprising providing a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof, and contacting the plasma polymerised polyoxazoline polymer film with the biomolecule or a derivative or precursor thereof under conditions to bind the biomolecule to the plasma polymerised polyoxazoline polymer film.

[0012] The oxazoline monomer may be a substituted oxazoline with a substituent at any of the 2-, 4- or 5-positions of the oxazoline ring or any combination of these substituents. In embodiments, the oxazoline monomer is selected from the group consisting of 2-substituted oxazolines, 4-substituted oxazolines, 5- substituted oxazolines, 2,4-disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, and 2,4,5-trisubstituted oxazolines. In some specific embodiments, the oxazoline monomer comprises a 2-substituted oxazoline. In specific embodiments, the oxazoline monomer is a 2-alkyl-2- oxazoline.

[0013] The conditions required to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer may comprise a power of from about 10W to about 50W, a deposition time of from about 1 minute to about 7 minutes, and/or a monomer pressure of from about 1.1 to about 3 x 10^"1 mbar. In embodiments, the conditions required to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer may comprise a power of greater than 30W for a time of greater than 5 minutes.

[0014] In embodiments, the plasma polymerised polyoxazoline polymer film has a thickness of greater than 3()nm.

[0015] A range of substrate materials can be coated by plasma deposition. Suitable substrate materials include glass, silicon, metals, plastics, polymeric materials, biomaterials, surfaces comprising biological molecules, surfaces comprising small organic molecules, surfaces comprising inorganic molecules, etc. In embodiments, the substrate is glass. In other embodiments, the substrate is silicon. In still other embodiments, the substrate is gold. In other embodiments, the substrate is a particle, such as a nanoparticle, drug particle or particle comprising a biological molecule.

[0016] The biomolecule of the fifth and sixth aspects of the invention can be any biomolecule that can be attached to the surface of the substrate, such as any biomolecule having a carboxylic acid group.

The biomolecule may for example be selected from amino acids, peptides, proteins, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents,

antithrombogenic agents, and cell attachment proteins. Examples of such biomolecules include growth factors such as endothelial cell growth factor, epithelial cell growth factor, osteoblast growth factor, fibroblast growth factor, platelet derived growth factor, neural growth factor, or angiogenin growth factor; antimicrobial agents such as lysozyme or penicillin; antithrombogenic agents such as heparin, fractionated heparins (eg., on an AT-III column), heparan, heparan sulfate, chondroitin sulfate, modified dextran, albumin, streptokinase, tissue plasminogen activator (TPA) or urokinase; cell attachment proteins such as fibronectin or laminin; thrombogenic agents such as collagen or a hydrophilic polymer such as hyaluronic acid, chitosan or methyl cellulose, carbohydrates and fatty acids. The biomolecule may be in the form of particles or nanoparticles comprising the biomolecule.

[0017] The substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof that is used in the fifth and sixth aspects of the invention can be formed according to the method of the third aspect of the invention.

BRIEF DESCRIPTION OF DRAWINGS

[0018] Embodiments of the present disclosure will be discussed with reference to the accompanying drawings wherein: [0019 ] Figure 1 shows plots of plasma deposited POx films film thicknesses as measured by ellipsometry for a range of substrate, exposed or not to water, acid, base and salt solution. A. high polymer flow variable deposition times b. low polymer flow, 7min deposition;

[0020 ] Figure 2 shows water contact angles on plasma deposited POx thin films;

[0021 ] Figure 3 shows XPS analysis of films deposited with low monomer flow (1.1 10- 1 mbar);

[0022 ] Figure 4 shows FTIR spectra of representative Plasma deposited POx thin films, deposited at low monomer flow for 7min at 10W (red), 30W (purple) and 50W (blue);

[0023 ] Figure 5 shows qualitative visual evidence of gold nanoparticles binding on POx plasma coated substrate and control AA substrate, subjected to different washing steps;

[0024] Figure 6 shows gold atomic concentration in% for AA, 30W7min, 50W7min samples after rinsing with water, SDS lv%, NaCl 5M, or NaOH pHIO solutions;

[0025] Figure 7 shows albumin adsorption study via QCM measurement for allylamine coated reference sample (top) and Pox 50W coated sample (bottom);

[0026] Figure 8 shows QCM analysis of protein adsorption on plasma deposited POx film with initial incubation in water (top), or polyacrylic acid (bottom);

[0027] Figure 9 (a) shows the thickness of samples as detennined by ellipsometry for varying polymerisation powers and times and (b) N:C ratios of POx films made by 5 minute polymerisation times as detennined by XPS;

[0028] Figure 10 shows eluted safranin as indication of total biofilm as a percentage of biofilm formed of plasma polymerised allylamine films ±standard error of mean;

[0029] Figure 11 shows microscope images of full biofilm grown of glass (left) and reduced\dislodged biofilm on plasma deposited POx (right) showing the difference between fouled and non-fouled samples;

[0030] Figure 12 shows biofilm growth on plasma deposited POx film in half treated 24-well tissue culture plate (left) and the interface of the coated\uncoated areas on the plate as observed via microscopy (right);

[003 1] Figure 13 shows photograph of stained S. epidermidis on glass, 50W 5 minute plasma deposited POx samples, P 1 , P2 and P3 (left to right) with 50W pulsed plasma conditions listed below; [0032] Figure 14 shows normalised metabolic activity of HDF grown on ppAA and 10W, 20W, 40W and 50W plasma deposited POx films as an indication of total HDFs adhered to the surface after 3 days;

[0033] Figure 15 shows the effect of plasma deposition conditions on film stability as determined by loss of film thickness when incubated in aqueous environments;

[0034] Figure 16 shows low fouling properties of plasma polymerized oxazolines formed by polymerization of a) 2-Methyl-2-Oxazoline and b) 2-Ethyl-Oxazoline at 2.3x10^"1 mbar at various deposition powers as percentage of total surface coverage. Error bars show ±SEM;

[0035] Figure 17 shows bacteria grown on a) untreated coverslip b) plasma polymerized 2-Methyl-2- Oxaoline and c) 2-Ethyl-2-Oxazoline deposited at 50W;

[0036] Figure 18 shows low fouling properties of plasma polymerized oxazolines formed by polymerization of a) 2-Methyl-2-Oxazoline and b) 2-Ethyl-Oxazoline at 50W with various deposition pressures shown as percentage of total surface coverage. Error bars show ±SEM; and

[0037] Figure 19 shows bacteria grown plasma polymerized 2-Methyl-2-Oxaoline deposited a) at 1.6 xl O^"1 mbar and b) 2.9 xlO^"1 mbar at 50W. Bacteria grown plasma polymerized 2-Ethyl-2-Oxaoline deposited c) at 1.6 x l O^"1 mbar and d) 2.9 xlO^"1 mbar.

DESCRIPTION OF EMBODIMENTS

[0038] Provided herein is a plasma polymerised polyoxazoline polymer. Also provided herein is a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof.

[0039] As used herein, the term "polyoxazoline" means a homopolymer or copolymer formed from at least one oxazoline starting material or monomer. The polyoxazoline polymer may or may not comprise intact oxazoline moieties. The polyoxazoline polymer may be a copolymer formed by plasma polymerisation of at least one oxazoline starting material or monomer and at least one comonomer. The comonomer may be chosen based on the desired properties it may provide to the polyoxazoline polymer and/or its suitability for plasma polymerisation (e.g. its vapour pressure or volatility). The comonomer may be selected from the group consisting of but not limited to: silanes, siloxanes, fluorocarbons, hydrocarbons, reactive functional monomers, organo-based monomers, and unsaturated monomers such as N-vinylpyrrolidone, hydroxyethylmethacrylate, acrylamide, dimethylacrylamide,

dimethylaminoethylmethacrylate, acrylic acid, methacrylic acid, a vinyl substituted polyethylene or polypropylene glycol, a vinylpyridine, and a vinylsulfonic acid. [0001 ] The plasma polymerised polyoxazoline polymer and polymer film on a surface of a substrate can be prepared by exposing the surface of a substrate to a plasma comprising an oxazoline monomer vapour under conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer on the surface of the substrate.

[0040] The conditions required to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer may comprise a power of from about 10W to about 50 W, a deposition time of from about 1 minute to about 7 minutes, and/or a monomer pressure of from about 1.1 to about 3 x l O^"1 mbar. A power of greater than 30W for a time of greater than 5 minutes are particularly suitable conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer because they provide stable plasma polymerised polyoxazoline polymer films having a thickness of greater than about 3()nm.

[0041 ] The plasma comprising an oxazoline monomer vapour is formed at reduced pressure in a vacuum chamber. Thus, the step of exposing the surface of a substrate to a plasma comprising an oxazoline monomer vapour may include placing the substrate in a chamber, sealing the chamber, forming a plasma in the chamber, introducing a vapour containing the oxazoline monomer into the chamber, and maintaining the substrate at a temperature suitable for polymerisation of the oxazoline monomer so as to form a polymer film on the surface. Persons skilled in the art will understand that a plasma is an electrically-excited ionised gas or gases, that, upon excitation (eg. ignition), forms a highly reactive environment that can modify materials directly exposed to the plasma discharge. The plasma deposition step can be operated over a wide range of pressures (for example, from 10 mTorr to above atmospheric pressure (eg. lOx atmosphere or higher)). The plasma may consist of a combination of an inert gas (eg. helium, neon, argon, krypton, xenon, radon) and the oxazoline monomer. The plasma can be formed at a range of frequencies (low-frequency direct current (DC) and alternating current (AC), pulsed DC, radio frequency (RF), and microwave).

[0042] Plasma polymerisation is a niche technique for creating polymer thin films [26, 27]. The method is simple, versatile and environmentally friendly. It enables the formation of nanometre thin coatings on any type of solid substrate without any substrate preparation required (typically required by other methods for surface modification) and without using organic solvents. A number of oxazoline ring containing compounds are low molecular weight liquid at room temperature (from 85 g.mol ¹ for 2- methyl oxazoline to 147 g.mol^"1 for 2-phenyloxazoline) which makes them perfectly suitable candidates for plasma polymerisation.

[0043 ] The oxazoline monomer may be a substituted oxazoline with a substituent at any of the 2-, 4- or 5-positions of the oxazoline ring or any combination of these substituents. Any of these oxazolines can be used to form the plasma polymerised polyoxazoline polymer provided they are a vapour under the plasma deposition conditions used. In embodiments, the oxazoline monomer is selected from the group consisting of 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2,4- disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, and 2,4,5- trisubstituted oxazolines. The substituent(s) on the oxazoline ring may be selected from the group consisting of: halogen, OH, N0₂, CN, NH₂, optionally substituted Ci-C]₂alkyl, optionally substituted C₂- Ci₂alkenyl, optionally substituted C₂-Ci₂alkynyl, optionally substituted C₂-Ci₂heteroalkyl, optionally substituted C₃-Ci₂cycloalkyI, optionally substituted C₂-Ci₂heterocycloalkyl, optionally substituted C₂- Ci₂heterocycloalkenyl, optionally substituted C₆-Ci₈ aryl, optionally substituted Ci-Ci₈heteroaryl, optionally substituted Ci-C_]2alkyloxy, optionally substituted C₂-C_]2alkenyloxy, optionally substituted C₂- Ci₂alkynyloxy, optionally substituted C₂-Ci₂heteroalkyloxy, optionally substituted C₃-Ci₂cycloalkyloxy, optionally substituted C₃-C_]2cycloalkenyloxy, optionally substituted Ci-C_]2heterocycloalkyloxy, optionally substituted C₂-C_{] 2}heterocycloalkenyloxy, optionally substituted C₆-Ci₈aryloxy, optionally substituted Ci-Ci₈heteroaryloxy, optionally substituted Ci-C₁₂alkylamino, S0₃H, S0₂NH₂, S0₂R, SONH₂, SOR, COR, COOH, COOR, CON RR , NRCOR , NRCOOR, NRS0₂R^', N RCON R^'R^", and NRR'. In some embodiments, the oxazoline monomer comprises a 2-substituted oxazoline. In specific

embodiments, the oxazoline monomer is a 2-alkyl -2-oxazo line. The alkyl substituent may be a C]-Ci₂ alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, etc.

[0044] In specific embodiments, the oxazoline monomer is selected from the group consisting of 2-alkyl - 2-oxazolines and 2-aryl-2-oxazolines. The alkyl substituent of the 2-alkyl -2-oxazolines may be a C] -C₁₀ alkyl, such as methyl, ethyl, n-propyl, i-propyl, n-butyl, i-butyl, t-butyl, pentyl, and the like. The alkyl substituent may be optionally substituted. The aryl substituent of the 2-aryl-2-oxazolines may be a C5-C₁₀ aryl, such as optionally substituted phenyl, optionally substituted naphthyl, optionally

substituted thienyl, optionally substituted indolyl, and the like.

[0002] The plasma polymerised polyoxazoline polymer film can be deposited onto any substrate. The substrate may be a solid substrate, particles, nanoparticles, powders, etc. Suitable substrate materials include glass, plastics, ceramics, silicon, organosiloxanes, paper, paper laminates, cellulose, carbon fibre, metals, rubber, biomaterials, surfaces comprising biological molecules, surfaces comprising small organic molecules, surfaces comprising inorganic molecules etc. The plastic may be selected from the group consisting of: polycarbonate, polyethylene, polypropylene, polystyrene, polytetrafluoroethylene, polyethylene terephthalate; polyethylene naphthalene dicarboxylate, tetrafluoroethylene- hexafluoropropylene copolymers, polyvinyl-difluoride, nylon, polyvinylchloride, copolymers of the aforementioned, and mixtures of the aforementioned. In embodiments, the substrate is glass. In other embodiments, the substrate is silicon. In still other embodiments, the substrate is gold. [0003] The substrate may be in the form of particles of any of the aforementioned materials.

Alternatively, or in addition, the particles may be formed from nanoparticles, drug particles or particles comprising a biological molecule.

[0004] The surface of the substrate may be treated prior to deposition of the plasma polymerised polyoxazoline polymer. For example, the surface may be treated by cleaning with a detergent, water or a suitable solvent. Alternatively, or in addition, the surface may be treated by exposing the surface to air in a plasma chamber in order to activate the surface.

[0045] Polyoxazoline (POx) based polymers formed using classical solution chemistry techniques have shown many interesting properties useful in delivery of therapeutics [9]. For example, POx has been conjugated with peptides and enzymes for improved drug delivery. Functionalisation of drug carriers, such as liposomes, by POx has also been shown to provide prolonged and/or targeted drug release. The thermoresponsiveness of POx derived polymers and their self-assembly behaviour in solution have also attracted much attention [10].

[0046] Despite the relatively large number of studies related to the formation and uses of polyoxazolines in solution, reports on their grafting to surfaces or organisation in thin films are not as common. This is most likely due to the fact that polyoxazoline immobilisation onto surfaces using classical solution chemistry is a tedious process, in which POx need to be initially synthesised in bulk solution before being grafted onto a substrate in a second step. So far, the different techniques which have been employed to generate POx coated surfaces include spin coating, grafting to, grafting from, photopolymerisation and electrostatic interactions. Since the initial work of Konradi and co-workers in 2008 on polyoxazoline bottle brush brushes [ 16, 17], these surfaces have been generated by several other groups for investigation of their passive non- fouling and antimicrobial properties [ 1 1, 18-20]. The general consensus so far seems to be that the nature of the monomer and in turn the wettability of the coatings tends to control their anti- fouling efficiency. Indeed, hydrophilic poly-2-methyl-2-oxazoline (PMeOx) supresses protein [ 18], cell [ 19 ] and bacteria [ 1 1 ] adsorption while more hydrophobic poly(2-ethyl-2-oxazoline)(PEtOx) [21 ] and poly-n-propyl-2-oxazoline (PnPrPOx) [ 19] promotes cell growth. Beside anti-fouling functions, POx coated surfaces are slowly being studied for new area of application such as biosensing [ 22 ].

[0047] Solution phase formation of polyoxazolines relies on opening of the oxazoline ring. Yet, for selected biomedical applications, such as antibody binding for biosensing purposes, it would be beneficial to retain the oxazoline ring to act as a reactive agent in selective biomolecule binding. The reactivity of the oxazoline ring present on the co-terminus of polyoxazolines has been used for conjugation with protein and drugs in solution [23 ]. The reactivity of the oxazoline ring is believed to lead to the formation of a covalent amide bond by reaction with a carboxylic acid function [24, 25]. Oxazoline based coatings deposited by plasma polymerisation offer an attractive alternative to conventional methods. The reason for this is the ease of coating preparation and the capacity to deposit on any type of substrate material. Furthermore, plasma deposited coatings may allow for the retention of intact oxazoline rings at the surface, which is typically not the case when other techniques for surface preparation are used. Retention of such reactive chemical functionalities is of interest for a number of applications in the biomedical space since it allows convenient and rapid covalent coupling of proteins, antibodies and other ligands or nanoparticles that carry carboxylic acid groups.

[0048 ] POx have also been shown to have anti-fouling properties and their efficiency at repelling proteins and bacteria has been shown to be equivalent to the gold standard PEG (polyethylene glycol) [ 1 1, 12]. Therefore, according to a fourth aspect there is provided a substrate comprising an antibiofouling surface, wherein the antibiofouling surface comprises a plasma polymerised polyoxazoline polymer.

[0049 ] We have shown that ligands and biological molecules can be immobilised on substrates comprising a plasma polymerised polyoxazoline polymer film on a surface thereof. Therefore, according to a fifth aspect, there is provided a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof and one or more ligands and/or biomolecules bonded to the polyoxazoline polymer film.

[ 0050] According to a sixth aspect, there is provided a process for immobilising a biomolecule on a surface of a substrate, the process comprising providing a substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof, and contacting the plasma polymerised polyoxazoline polymer film with the biomolecule or a derivative or precursor thereof under conditions to bind the biomolecule to the plasma polymerised polyoxazoline polymer film.

[0051 ] The biomolecule can be any biomolecule that can be attached to the surface of the substrate, such as any biomolecule having a carboxylic acid group. The biomolecule may for example be selected from amino acids, peptides, proteins, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents, antithrombogenic agents, and cell attachment proteins. Examples of such biomolecules include growth factors such as endothelial cell growth factor, epithelial cell growth factor, osteoblast growth factor, fibroblast growth factor, platelet derived growth factor, neural growth factor, or angiogenin growth factor; antimicrobial agents such as lysozyme or penicillin;

antithrombogenic agents such as heparin, fractionated heparins (eg., on an AT-II1 column), heparan, heparan sulfate, chondroitin sulfate, modified dextran, albumin, streptokinase, tissue plasminogen activator (TPA) or urokinase; cell attachment proteins such as fibronectin or laminin; thrombogenic agents such as collagen or a hydrophilic polymer such as hyaluronic acid, chitosan or methyl cellulose, carbohydrates and fatty acids. The biomolecule may be in the form of particles or nanoparticles comprising the biomolecule. [0052 ] Advantageously, the step of contacting the plasma polymerised polyoxazoline polymer film with the biomolecule or a derivative or precursor thereof does not require the use of a coupling agent, such as carbodiimide. The biomolecule may be bound to the plasma polymerised polyoxazoline polymer film by one or more covalent bonds.

[0053] The substrate comprising a plasma polymerised polyoxazoline polymer film on a surface thereof that is used in the fifth and sixth aspects of the invention can be formed according to the method of the third aspect of the invention.

EXAMPLES

[0054] Example 1 - Preparation and properties of plasma polymerised polyoxazoline coatings [0055] Materials

[0056] 2-Methyl-2-oxazoline, polyacrylic acid 36%, NaOH pellets, acetic acid, NaCl, SDS 10v%, Phosphate Buffer Saline (PBS) tablets, Streptavidin, Bovine Serum Albumin, and common solvents were purchased from Sigma Aldrich and used without further purification. Goat anti-human podocalyxin anti bodies were purchased from alpha diagnostic international. Hydrogen tetrachloroaurate (99.9985%, ProSciTech), trisodium citrate (99%, BHD Chemicals, Australia Pty. Ltd.), and 2-mercaptosuccinic acid (97%, Aldrich), were used as received. Q-Sense Gold QCM sensors w^rere purchased from ATA scientific. Ultra high purity water obtained from a MilliQ system with resistivity greater than 18MQ.cm, was used for all experiments and rinsing steps.

[0057] Plasma polymerisation

[0058] Plasma polymerisation was performed in a custom built capacitively coupled bell-chamber reactor. Solid substrates (glass coverslips, silicon wafers and QCM crystals) were cleaned with acetone and ethanol and dried with nitrogen flow. Clean substrates were added to the plasma reactor and the chamber brought to a vacuum. A three minute air plasma was used to further clean and prime the samples. When the chamber reached a base pressure of 3.5x1 ()^"2mbar the 2-methyl-2-oxazoline monomer was introduced via a needle valve until the desired monomer flow was achieved and the working pressure in the chamber steady (2.3xl0^_1 mbar and l . lxlO^"1 mbar for "high" and "low" flow samples respectively). Plasma was ignited using RF powers varying from 10 to 50 W in continuous mode. The monomer deposition time was varied from 1 to 7 minutes.

[0059] Fabrication of COOH-Gold nanoparticles and binding study. [0060] AuNPs were synthesized following established methods previously described [28]. Briefly nanoparticles 15nm in diameter were prepared by citrate reduction of HAuCl₄. The carboxylic acid functionality was provided via surface modification with 2-mercaptosuccinic acid. After rinsing with MilliQ water, plasma coated glass coverslips were incubated with a suspension of gold nanoparticles for 6h in 24 well plates. The gold nanoparticle suspension was then aspirated and the coverslips rinsed 3 times with MilliQ water. SDS 10v% or 5M NaCl solution were then added and left for lh. Finally, the substrate was thoroughly rinsed with flowing MilliQ water and dried with nitrogen flow.

[0061 ] PAA blocking

[0062 ] Plasma coated glass coverslips were initially rinsed with MilliQ water before incubating with lw% polyacrylic acid (PAA) solution for lh in 24 well plates. After aspirating the PAA solution, the substrates were rinsed 3 times with MilliQ water, before washing with SDS 10v%, or 5M NaCl solution as described above. Finally all samples were thoroughly rinsed with large amounts of MilliQ water and dried with nitrogen flow.

[0063 ] Protein adsorption-QCM

[0064 ] Quartz Crystal Microbalance with Dissipation monitoring (QCM-D) is a surface sensitive technique used to evaluate changes in thin films mass and viscoelastic properties. It was used to assess, in real time, the plasma polymer thin films interaction with proteins in a physiological fluid environment under flow conditions. QCM-D measurements were performed with a Q-Sense E4 instrument (Q-Sense, Sweden) offering sensitivity in the ng/cm² range. POx plasma polymer films were deposited onto gold QCM sensors and tested in parallel with two control sensors coated with allylamine (AA) plasma polymer films. The fluid flow over the sensors was set to 0.1 mL mm ¹ using a variable-speed peristaltic pump (ISM 935, 1DEX Health & Science, and Germany) and kept constant for the whole experiment. All four sensors were initially equilibrated in MilliQ water, and subsequently in PBS, until both dissipation and frequency trace stabilised. The sensors were then exposed to 0.1 mg.mL ¹ protein solution (BSA, fibronectin, SAV or podocalyxin) for 90min, before rinsing with PBS 15min, washing with SDS 10% for 15min, and further rinsing and re-equilibrating in PBS for a minimum of 30 min or until the trace stabilised.

[0065] XPS

[0066] XPS analysis was conducted on a SAGE X-ray photoelectron spectrometer with a

monochromatic Mg radiation source operating at 15kV and 10mA. Survey spectra acquired at a pass energy of 12()eV over the energy range 0-1 lOOeV, and with a resolution of leV were used to determine the atomic composition of the films. The chemical state of nitrogen and carbon atoms were investigated from high resolution spectra recorded at 20eV pass energy. The charge compensation effects were corrected by calibrating all spectra to the neutral carbon peak at 285eV. CasaXPS software was used for spectra analysis.

[0067] Ellipsometry

10068] Plasma polymer film thickness measurements were conducted with an imaging ellipsometer A J.A Woolam Co. Variable Angle Spectroscopic Ellipsometer (VASE). Data were analysed using

WVASE32 software.

[0069] Contact angle

[0070] Static advancing and receding contact angles of water (MilliQ, p = 10³ kg m^"3, η = 8.9 ^■ l O^"4 Pa s) were measured on plasma polymer coated silicon wafers using a sessile drop apparatus and OCA, SCA20, dataphysics software. All measurements were conducted on a specially isolated, vibration free bench. Reproducible static contact angle data were obtained from a minimum of five measurements on each sample.

[0071] ETIR

[0072] Fourier Transform Infra-Red spectroscopy (FTIR) was conducted with a Nicolet spectrometer. For FTIR analysis purposes, 50mg of potassium bromine (KBr) powder was coated in the same plasma reactor and using the same experimental conditions as the other solid substrates. The coated KBr powder was analysed using a smart diffuse reflection accessory. All spectra were collected at room temperature over the spectral range 65(⁾-4000cm^"1 with a resolution of 4cm^"1 and 512 scans. Omnic software was used for spectra analysis.

[0073] Results and discussion

[0074] 2-Methyl-2-oxazoline was used as a precursor for plasma polymerisation. In a tailor made plasma reactor, polyoxazoline (POx) plasma polymer films were formed on solid substrates using powers ranging from 10 to 50W, deposition times ranging from 1 to 7 min and monomer pressure of 1.1 and 2 x 10^"1 mbar.

[0075] The thickness and stability of the plasma deposited POx films were dependent on the plasma RF power, monomer deposition time and precursor flow rate. Ellipsometry measurements of film thicknesses are summarised in Figure 1. Using different deposition conditions the POx plasma films can be varied in a controlled fashion. When the monomer pressure was 2 x 10^"' mbar, film thicknesses ranging from 20 to 76 nm were formed, with film thickness increasing with deposition time going from 1 to 5 minutes, and RF power from 10 to 50 W (Figure l a). Notably, the films formed with lower RF power showed significant thickness losses after extended exposure to water. A 90% thickness loss was, for example, recorded for the 10 W 5min films. Using lower monomer pressure, with longer deposition times enables the formation of films ranging from 30 to 60nm thick, with increased stability in not only water but also, salt solution (5M NaCl, artificial urine), acid (CH₃COOH, pH 4) and base solution (NaOH, pH 10), as can be seen in Figure lb. In particular, films obtained using the higher RF powers (30W and above) exhibit very good stability to water, with no film thickness loss, even after 24h exposure (dark blue bars). The film formed at 20W RF power did show a 20% thickness loss within the first hour of exposure to water and no significant further loss was observed after 24h soaking, thus indicating that film matter loss caused by water occurs very rapidly after exposure, and may well correspond to the desorption of loose monomer fragments only physically deposited on the substrate at the end of the plasma polymerisation process from species remaining in the chamber after the cross linking RF power has been turned off.

[0076 ] The stability of the plasma deposited POx films was further confirmed via static contact angle measurements. Contact angle measurements were conducted on dry POx films, films rinsed with water, and films incubated in MilliQ water, acid and basic solutions for l h. All films studied were hydrophilic with advancing contact angles below 90°. The water contact angle measured on plasma deposited POx films appeared to be an increasing function of the RF powers, as shown in Figure 2. For low power films, typically below 30W, a significant increase in water contact angle occurs immediately after substrate exposure to water. This initial change is not further increased by prolonged exposure to water (data not shown) or incubation in salt solution. The water contact angle of higher power polymer films is not affected by plasma deposited POx films incubation in salt solution, acid or base (data not shown) thus supporting the stability observation made via ellipsometry measurements.

[0077] The difference in intrinsic wettability for films formed using different plasma RF powers (see Figure 2a for example), suggests that the chemistry of the films differs depending on the coating deposition conditions. This is not unexpected for plasma polymer thin films. Indeed, the structural fragmentation and recombination of the precursor molecule occurring during plasma polymerisation inevitably leads to a complex thin film chemistry which is known to be strongly dependant on the plasma power and monomer flow used. Typically, reactive functional groups tend to be lost by high power plasma polymerisation while gentler conditions are more likely to enable the retention of the precursor chemical functionality. In the case of 2-methyl-2-oxazoline, it is of particular interest to assess whether or not the oxazoline ring is retained throughout the plasma polymerisation process. Although only very few experimental data on the wettability of polyoxazoline film have been released, the work of Zhang el al on poly(2-oxazoline) bottle brush brushes (BBB) provides insight [19]. Poly-2-oxazoline BBB, featuring an open ring configuration (PMeOx) have a water contact angle of 38°, while PiPox (poly-2-isopropenyl-2- oxazoline) brushes were reported to have a contact angle of 51 °.

[0078] The atomic compositions of the plasma deposited POx films were determined by XPS. A typical survey spectrum is shown in Figure 3a, and the detailed atomic composition for the range of polymer deposited with low polymer flow is shown in Figure 3b. The nitrogen content slightly decreases with plasma power from 18% at 10W to 15% at 50 W. A similar trend is observed with films deposited with high monomer pressure, but the nitrogen content is universally higher on these samples (from 21 % to 19%). The nitrogen to oxygen ratio remains relatively constant over the range of substrates investigated and always higher than that of the monomer. Analysis of the high resolution C 1 s spectra (Figure 3 c) revealed the presence of about 30% of C-0 environment, with these quantities slightly decreasing from 35% for 10W film down to 27% for 50W films deposited with low monomer flow, as shown in Figure 3d. Here again, the same trend is observed for the films deposited with higher monomer flow and the total amount of C-0 environment is a slightly higher on these films. Interestingly, the amount of C-0 environment consistently decreases after film exposure to water ( lh soaking in MilliQ, follow by drying with nitrogen gas). This is the case for all samples except the film deposited at l OW which appeared to delaminate after exposure to water.

[0079 ] The IR spectra of the plasma deposited POx films are shown in Figure 4. All plasma deposited POx films studied present a broad -OH stretching bands between 3200 and 3400 cm^"1 originating from substrate hydration. The peak observed at 2950 corresponds to sp² and sp³ C-H stretching. A very intense band is observed for all samples at 2170 cm^"1 with a shoulder at 2250 cm^"1 which are attributed to alkyne C≡C and/or isocyanate 0=C=N and nitrile C≡N liaison respectively. The strong and rather wide band observed around 1657 cm^"1 is associated with the stretching of the C=N bond present in the oxazoline ring. However the asymmetric shape of this peak suggests that an overlap occurs with the C=0 stretching band expected, for the amide function of PMeOx, to be at 1627 cm^"1 [ 19]. At low power, the C=0 band is resolved but the intensity ratio between these two signal quickly decrease as the power increases, indicating that the chemistry of the plasma deposited POx films is dependent on the RF powers. The medium band present at 1370 cm^"1 is attributed to C-H bending modes, while the strong band at

1 130 cm^"1 is assigned to the alkoxy C-0 bond, also present in the oxazoline ring. The intensity of this band also varies significantly with the plasma power and is stronger for 10W samples. In PMeOx, typically without oxazoline rings, the C-0 band is generally absent from the FTIR spectrum. Finally a sharp band at 800 cm^"1 is present for some but not all of the plasma deposited POx films. Aromatic ring skeletal vibrations are typically found in this wavelength range, although former studies claimed that the oxazoline ring skeletal vibration occurs at high wavelength (950 cm^"1) [29].

[0080] Overall, the FTIR and XPS chemical analysis of the plasma deposited POx films reveal a very complex film chemistry consisting of a variety of reactive chemical functions whose presence/absence and concentration varies with the plasma deposition conditions used. In particular, in view of the present results, the retention of the oxazoline ring cannot be ruled out.

[0081] In order to investigate the reactivity of POx plasma polymer films with carboxylic acid chemical groups, glass coverslips coated with plasma deposited POx were incubated with COOH-functionalised gold nanoparticles in 24 well plates. 16nm gold nanoparticles used in this experiment absorb light at about 520 nm due to surface plasmon resonance phenomena. As a result, the gold nanoparticles appear pinkish in colour. Thus, a pinkish colour of the substrate is indicative of gold nanoparticle adsorption on the otherwise transparent glass coverslips.

[0082 ] The nature of the bond between the COOH functionahsed gold nanoparticles and the POx coating was challenged by several rinsing and washing steps with water, salt (1M, 3M, and 5M NaCl), surfactant (SDS lv%), acid and base solutions. Allylamine ("AA") coated substrates, on which gold nanoparticle adsorption is expected to be driven by electrostatic interactions, were used as control surfaces.

[0083 ] The visual proof of gold nanoparticle binding on POx thin films deposited at 10, 30, and 50W, for 5 min at high monomer flow, is shown in Figure 5. From the intensity of the pink coloration it appears that the gold nanoparticles did not adsorb on the 1 OW substrates. This is not surprising in view of the poor stability of the films already evidenced via contact angle, ellipsometry and XPS analysis. On the other hand, both 30W and 50W adsorbed gold nanoparticles as well as the control AA coated substrate.

Equivalent experiments were conducted with plasma deposited POx films deposited for 7min at high monomer flow and returned similar results: in all cases, samples deposited at high power (typically above 30W and above) successfully bind gold nanoparticles. Also, for all POx substrates investigated, it is visually not possible to observe any clear changes in coloration between the various samples and after the different washing steps. In contrast, gold nanoparticle release in solution was observed when washing the AA coated samples with SDS and concentrated NaCl salt solutions.

[0084] In order to quantitatively measure gold nanoparticle binding and desorption the surfaces were analysed using XPS. The poor stability of the 10W films was once again confirmed, with nitrogen atomic percentage decreasing from 19 to 1% after 6h incubation in the gold nanoparticle solution.

[0085] In Figure 6, we report the atomic percentage of gold for control AA coated substrate, and POx 30W and 50W deposited this time at high monomer flow rate for 7 minutes. The practical observation of gold nanoparticles being removed from AA surfaces by NaCl and SDS washes is confirmed by the XPS measurements, where, for example, a 50% loss of adsorbed gold is recorded after SDS wash and 25% after salt wash. This result indicates that some of the gold nanoparticles binding on the AA samples are doing so via reversible electrostatic interactions which are readily disrupted by exposure to surfactants and concentrated salt solutions. On the contrary, the binding of gold nanoparticles to POx surfaces does not appear to be affected by salt or pH. Interestingly, a slight decrease in the amount of gold nanoparticle is observed after SDS rinse for 50W samples, while in this condition the nanoparticle binding is unaltered on the 30W films which suggests a strong covalent binding. In the case of the 50W films, this could be that two different adsorption mechanisms are at play, namely electrostatic adsorption and covalent irreversible binding. These results indicate that the reaction of POx films with COOH -groups result in irreversible nanoparticle adsorptions.

[0086] The ability of plasma deposited POx films to bind protein was then examined via real time QCM- D measurements.

[0087] To date biomaterial used for proteins, and more specifically, antibody binding made via plasma methods rely on the formation of covalent bonds between the amine groups present on the biomolecules and carboxylic acid or aldehyde groups grafted to the biosensing platform [27]. Biomaterial conjugation with biomolecules typically involves several surface modification steps , including carboxylic acid activation, followed by primary protein covalent binding via carbodiimide coupling (eg. streptavidin), before the actual capture anti-body can be grafted to the substrate (eg. streptavidin antibody.) In contrast, the unique chemistry of the plasma deposited POx films comprising oxazoline ring groups presents significant advantages for simple, single step protein or anti body binding, without catalysts being needed.

[0088] In order to evaluate the protein binding efficiency of POx plasma films, real time QCM-D experiments were conducted under flow conditions in physiological buffer. Exposure to 0.1 mg/mL albumin solution resulted, for both POx and control allylamine coated sensors, in a sharp decrease in frequency, providing evidence of mass increase due to protein coupling to both plasma polymer films (Figure 7). In all cases, the sharp initial drop in frequency is followed within minutes by a much slower negative change regime which eventually reaches a quasi-equilibrium state after 6()min. The sensors were then subjected to thorough in situ rinsing and washing steps, first with PBS, then with concentrated SDS ( 10%) and finally, again with PBS. Analysis of the frequency trace throughout these washing steps revealed differences in the protein binding behaviour depending on the nature of the plasma polymer film.

[0089] On one hand, after initial rinse with PBS, the frequency of AA coated sensors increases slightly, indicating some protein desorption from the surface. Furthermore, after SDS wash (which is used to detach any physically adsorbed proteins), and final equilibration in PBS the AA sensors recovered their initial frequency, revealing that all adsorbed protein has been washed off the sensor surface.

[0090] On the other hand, no frequency change is observed for POx coated samples following PBS rinse, and a permanent negative deflection remains after washing with SDS, thus providing evidence of irreversible and covalent protein binding on POx plasma films. [0091 ] The relationship between the frequency change and the quartz electrode mass is given by the Sauerbrey equation. For the Q sense e4 QCM-D apparatus, the relationship is[30 |:

CAf

Am =

n where Δ/^'is the measured frequency shift (Hz), C= 17.7 ng Hz^"1 cm^"2 for a 5 MHz quartz crystal and n, is the overtone number.

[0092] The average amount of Albumin (BSA) covalently bound to POx plasma film can be estimated to 70 + 7 ng.cm-². BSA molecules have a molecular weight of 66.5 kDa and occupy 37nm² when closely packed, thus the maximum theoretical packing density is 30()ng.cm^"2 [27] . The amount of protein bound to the POx plasma polymer film roughly corresponds to 25% coverage. This is appropriate for biosensor platforms because it allows sufficient spacing between protein molecules for conducting binding reactions. The protein binding ability of POx plasma deposited films does not appear to depend on the nature of the protein: adsorption of streptavidin, antipodocalyxin and fibronectin were also measured. The amount of fibronectin binding to POx plasma films is 80ng/cm². This value is significantly larger than what has been reported in the literature for poly-2-methyl-2-oxazoline coated surface (<6ng/cm²) [ 16, 19], thus indicating the increased potential of plasma deposited PMeOx for protein binding as compared to PMeOx films made via conventional methods.

[0093] In order to further investigate the protein binding mechanism with plasma deposited POx films, another set of QCM experiment was performed where two POx coated samples were initially exposed to poly( acrylic acid) (PAA), before the introduction of the protein. It appears that the samples exposed to PAA did not adsorb any proteins, as can be seen in Figure 8. This result is consistent with the hypothesis that chemical functionality present on the surface of the POx plasma polymer films, such as oxazoline rings, reacts with carboxylic acid groups. Blocking these reactive functional groups with PAA, suppresses the ability of the film to bind to the carboxylic acid groups present on the protein.

[00941 These results, together with those of FTIR, XPS, and PAA binding, suggest that at least a small amount of oxazoline rings are retained during the plasma polymerisation process.

[0095] Conclusions

[0096 ] For the first time we report the plasma polymerisation of 2-substituted-2-oxazolines. Stable plasma deposited oxazoline films are created with thicknesses varying from 30 to 75nm depending on the deposition time and RF power used for plasma ignition. The chemistry of the hydrophilic films formed is rich and complex, as evidenced by XPS and FTIR analysis. Our results show that several different functional and reactive chemical functions are formed during plasma polymerisation (amide, isocyanate, carbonyl etc), and that the reactive oxazoline ring may be retained to some extent.

[0097] The reactivity of the plasma deposited POx films with carboxylic acids and proteins was shown. Real time QCM-D analysis revealed that irreversible protein adsorption occurs on the plasma deposited POx films, and that the surface coverages obtained are suitable for applications in biosensing.

[0098] Example 2 - Selective biological adhesion onto plasma polymerised POx surfaces

[0099] Undesirable bacterial adhesion to solid surfaces and the subsequent biofilm formation is a significant, unsolved problem in a number of areas [31,32]. Typical examples include biomedical devices [33,34], marine structures [31 ,32], membrane separation [35,36] and heat exchange systems [37]. Despite decades of extensive research a comprehensive solution to this problem has not been found.

[00100] The relevance of the problem is global and its consequences are costly. Infections caused by the bacterial colonisation of medical devices constitute more than half of all hospital acquired infections (HA1) and are the most costly and complex to treat [33,34]. Each year HAI cause

approximately 100,000 deaths in the USA at a cost to the healthcare system of about $40 billion annually [38 ]. In Australia, HAI cost the taxpayers over $ 1 billion in extra bed days alone [9]. It is also well- known that marine vessels have to be regularly cleaned from fouling caused largely by adhered microorganisms. The US Navy is estimated to spend $2.1 billion annually on problems related to biofouling [401. Filtration membranes in various industries need to be regularly replaced at great expense for the same reason. Undesirable bacterial adhesion is also a significant problem in the food processing industry [41 ] and food retailing [36], heating and cooling systems [42], liquid storage and transport [37], and many other industries [3 1 ].

[00101 1 A commonly used strategy to achieve biopassive antifouling properties is the addition of poly(ethylene) glycol (PEG) and PEG-copolymers. However, a common downfall with PEG based surfaces coatings is degradation [45-47]. For this reason alternatives have been investigated. One suitable substitute that recently gained considerable attention are the polyoxazoline based polymers (POx). A comparative review of the antifouling properties PEG and POx coatings by Konradi et al. [46] states that even with differences in surface architectures and test conditions within the literature, both surface types are typically equally effective in respect to antifouling ability. However, generally, POx surfaces show greater stability than PEG based surfaces generated in a similar manner.

[00102] So far POx have been prepared by wet processes which are tedious, costly and lengthy. In addition, these processes are conducted in organic solvents and need to be tailored for particular type of substrates. A process for generation of POx coatings that is suitable for preparation of strongly adherent coatings to the various type of substrates (for instance, medical implants are made from various types of materials eg metals, ceramics, polymers and composites) used in applications will be of enormous benefit.

[00103] Plasma polymerisation is a niche technique for creating polymer thin films [48,49]. The method is simple, versatile and environmentally friendly. It enables the formation of nanometre thin coating on any type of solid substrate without any substrate preparation required (typically required by other methods for surface modification) and without using organic solvents. A number of oxazoline ring containing compounds are low molecular weight liquid at room temperature (from 85g/mol for 2- methyl oxazoline to 147g/mol for 2-phenyloxazoline) which makes them perfectly suitable candidates for plasma polymerisation.

[00104] Oxazoline based coatings deposited by plasma polymerisation may become an attractive alternative to conventional methods. We therefore investigated the use of plasma polymerisation of 2- methyl-2 -oxazoline to generate substrate independent polyoxazoline thin films with tailored bio-adhesive properties.

[00105] Materials

[00106] 13mm diameter glass coverslips were used as test substrates for biological interactions.

Si-wafers were coated and used for all thickness measurements. 2-Methyl-2 -oxazoline (Sigma-Aldrich, Australia) was used as a base monomer. Acetic acid and poly(acrylic acid) (Sigma-Aldrich, Australia) was used for post modification of samples. S. epidermidis (ATCC 35984) and Human Dermal Fibroblasts (HDF) were used as models of biological interaction. Cold Filterable Tryptone Soya Broth (TSB: Oxoid Australia) was used as bacterial growth medium. Dulbecco's Modified Eagle Medium (DMEM: Gibco, Life Technologies, Australia), 10% v/v Fetal Bovine Serum (FBS, Ausgenex, Australia), 0.625 μg/mL amphotericin B (Sigma-Aldrich, Australia), 100 IU/mL penicillin and 100 mg/mL streptomycin (Gibco, Life Technologies, Australia) was used as HDF culture medium.

[00107] Sample preparation

[00108] Plasma polymerisation was performed in a purpose built capacitively coupled bell- chamber reactor based on previous designs. Substrates were cleaned by sequential acetone and ethanol washes and dried with nitrogen. Clean substrates were added to the plasma reactor and the chamber brought to a vacuum. A three minute air plasma was used to further clean and prime the samples. When chamber reached appropriate base pressure (3.5x10^"2mbar) the oxazoline monomer (2-methyl-2- oxazoline) was introduced via a needle valve until a steady working pressure of 2.3x10^"1 mbar was achieved. Plasma was ignited using various RF powers in both continuous and pulsed plasma modes. A subset of samples was treated with acetic acid or poly-acrylic acid (PAA) to look at changes in surface chemistry and effect on fouling.

[00109] Surface characterisation

[001 10] X-ray photoelectron spectroscopy (XPS) was used to confirm the presence of the plasma polymer and chemically characterise the thin films. Using a SAGE XPS fit with a monochromatic Mg source, a survey spectrum and C 1 s were taken 120eV and 20eV pass energy respectively. Data was processed with CasaXPS software (Version 2.3.14) and all charges were corrected relative to C-C at 285. OeV. Coating thickness was determined via Ellipsometry. A J.A Woolam Co. Variable Angle Spectroscopic Ellipsometer (VASE) was used for all measurements and data analysed using WVASE32 software.

[001 1 1 ] Effect on biofilm formation

[001 12] S. epidermidis was used as a model organism for biofilm formation. S. epidermidis was streak plated onto nutrient agar plates and grown overnight at 37°C. Single colonies were picked and incubated in TSB overnight. S. epidermidis was diluted to l x lO⁶ CFU\mL based on OD₆oonm- Substrates were added to a 24 well plate and 400μί S. epidermidis was added to each well. The bacteria was incubated overnight at 37°C and allowed to form biofilms. After incubation all samples were washed twice with MilliQ distilled water and 200μί of safranin stain added so that biofilms cold be visualised. Excess safranin stain was washed off of the samples before the stain was eluted with 33% glacial acetic acid in ethanol and quantified using a plate reader (Ab₄90nm)- Samples of different plasma conditions, ages and with different post treatments were analysed for antibiofouling effects.

[001 13] To confirm the substrate independence of the polymerisation technique, a 12-well tissue culture plate was coated. A simple cover-mask was created to ensure only half of individual wells were coated. Using the same biofilm staining protocol the strongly adhered biofilm was visualised

microscopically.

1001 14] Fibroblast attachment

[001 15] Human derived dermal fibroblasts were seeded at lxl O⁴ cells per well and grown in

DMEM+10% FBS supplemented with 0.625 μg\mL amphotericin B, lOOIUXraL penicillin and mg\mL streptomycin. Fibroblasts were allowed to attach to samples and after 3 days cell viability was measured using an alamar blue assay. A stock solution of 1 K^gVmL alamar blue was prepared in PBS and filter sterilised. Stock was diluted 1 : 10 with culture media and cells left to incubate with 500μΙ_, of the solution for another 4h. 200μΙ_, of solution was transferred in individual wells of a 96-well plate and fluorescent intensity was read using a plate reader (λεχ =544nm and λεηι =590nm).

[001 16] Results and discussion

[001 17] The thickness of the plasma deposited POx films were determined by ellipsometry.

Figure 9a shows the thicknesses of coatings deposited using RF power of 10, 20, 40 and 50W and deposition time of 1, 2 and 5 minutes. Both higher deposition power and time lead to thicker films which indicate that in the range of deposition conditions used constructive processes of film build up dominate. The chemical composition of the coatings was detennined by XPS. Figure 9b shows the N to carbon ratio of coatings deposited using RF power of 10, 20, 40 and 50 W for 5 minutes. Increased plasma power seems to lead to loss of nitrogen in the coatings which may be caused by the higher fragmentation of the monomer at higher power.

[001 18] S. epidermidis was used as a model organism to evaluate the capacity of the plasma deposited POx coating to resist bacterial colonisation. This organism was used because it is a very strong biofilm former. S. epidermidis is the organism most often associated with medical device infections. 1()⁶ of bacteria ware was allowed to interact with the plasma deposited POx coated substrates and form biofilms overnight. Samples were then washed twice to remove any loosely bound bacteria before staining the formed biofilms with safranin. Excess safranin stain was washed off of the samples to ensure that the only remaining stain was that absorbed by bacteria. Various samples based on differing plasma polymerisation conditions were assessed. During the washing stages the biofilm detached from samples prepared using certain conditions. This indicates a loose binding of the bacteria and extracellular matrix. Results were observed via microscopy and quantified by eluting the stain and quantifying using spectroscopy.

[001 19] Figure 10 shows the level of biofilm formed on plasma deposited POx films prepared using deposition power of 10W, 20W, 40W and 50W and deposition time of 1 , 2, and 5 minutes compared to the total biofilm on plasma polymerised allylamine (ppAA) films. ppAA and glass were used as controls. All samples were blanked relative to glass treated with no bacteria. The results show an evident decrease in the total biofilm on samples prepared at the higher powers, i.e. 40W and 50W. Little to no change was seen in samples prepared at 10W and 20W. The 50W samples decreased total biofilm by approximately 65% and the 40W samples reduced total adhered biofilm by 40-50%. The difference between a highly fouled glass surface and non-fouled plasma deposited POx surface can be easily observed in the microscopy images shown in Figure 1 1. It is striking that on the control surface (left) bacteria readily adsorb and proliferate, and form large colonies and extracellular matrix. In comparison, bacteria adsorb to the POx coating (left) but do not seem to grow and form biofilm. [00120] The chemical analysis of the coating by XPS (presented in Figure 9b) show that there are no significant differences between coatings prepared using different power. However, coatings deposited using higher power proved to be more resistant to bacterial colonisation. Higher power generally results in more crosslinked coatings which have been proven to have greater stability when exposed to solvents.

[00121 ] The results presented in Figure 11 were obtained using coatings deposited on glass substrate. Figure 12 shows an image of the stained biofilm (left) and a magnification of the

coated\uncoated interface (right) in a well of a 24-well tissue culture plate; where half of the well is coated with the 50W 5 minute plasma deposited POx. The images unambiguously demonstrate that the POx coated area (right hand side of each image) substantially reduces the level of bacterial colonisation evident by the lower intensity of red colour used to stain the biofilm. This experiment demonstrates that coatings of the same quality can be deposited in absolutely the same way on ceramic and polymeric substrates.

[00122 ] We also attempted to use plasma deposition in pulsed mode for preparation of the coatings. In general, plasma polymerisation in "pulsed" mode should allow a better retention of the original monomer chemistry as there is less fractionation of the monomer due to the "off period. Efficacy of using a pulsed plasma technique for anti-biofilm surface generation was explored by varying on\off times of 50 W 5 minute sampl es. Figure 13 shows the antifouling efficacy of the plasma deposited POx deposited with various pulsed plasma conditions.

[00123] Sample P I , P2 and P3 had "on" times of 1ms, 10.5ms and 20ms respectively, with a total cycle of 21ms. Untreated glass coverslips and 50W 5 minute plasma deposited POx were used as controls. Samples were treated with S. epidermidis and stained with safranin as in Figures 10 and 1 1. High levels of biofilm are visible on the glass sample and samples P I and P2. Sample P3 shows a high level of antifouling ability comparable to the original 50W 5 minute sample.

[00124] Biocompatibility is an important consideration when determining the appropriateness of a surface modification, especially for use within the medical field [51 ]. Primary human derived dermal fibroblast cells (HDF) are used as a model for potential cytotoxic effects in vitro as they are associated with wound healing [52-55]. An alamar blue assay was used to quantify fibroblast metabolic activity relative to ppAA, as shown in Figure 14. This was used as a way to quantify the number of cells adhered to the glass, ppAA and 10W, 20 W, 40 W and 50W 5 minute plasma deposited POx samples. This assay assumes that each cell has the same metabolic activity. Samples produced at 10W and 20W show a reduction in HDF adhesion, as determined by viability. Interestingly, an increase in the HDF viability is seen as the plasma power increases. The 40W plasma showed similar viabilities as the untreated glass surfaces and the 50W samples showed similar viabilities as the ppAA surfaces which are known to provide an excellent surface for cell culture. [00125] Example 3 - Selective biological adhesion onto plasma polymerised POx surfaces

[ 00126] Sample preparation

[00127] Clean substrates were added to the plasma reactor and the chamber brought to a vacuum.

Oxazoline (2-methyl-2-oxazoline or 2-ethyl-2-oxazoline) precursors were deposited into the chamber via a needle valve until steady working flow rates were achieved. Plasma was ignited using various RF powers in a CW mode for selected times.

[00128] Surface stability

[00129] Figure 15 shows polymer thicknesses before and after incubation in aqueous medium as determined via EUipsometry. A J.A Woolam Co. Variable Angle Spectroscopic Elhpsometer (VASE) was used for all measurements and data analyzed using WVASE32 software.

[00130] Bacteria interaction

[00131 ] S. epidermidis was streak plated onto nutrient agar plates and grown overnight at 37°C.

Single colonies were picked and incubated in TSB overnight. S. epidermidis was diluted to lxl 0⁶ CFU\mL based on turbidity (OD6()0nm). Substrates were added to a 24 well plate and 400 xL S.

epidermidis suspension was added to each well. The bacteria was incubated overnight at 37°C and allowed to form biofilms. After incubation all samples were washed twice with MilliQ water to remove any loosely bound biofilm. 200 μΕ of safranin stain was added so that biofilms could be visualized. Excess safranin stain was washed off and samples imaged via microscopy. The results are shown in Figures 16 to 19.

[00132] In summary, preparation of POx coatings by plasma polymerisation has been carried out for the first time. The coatings have controllable thickness at the nanoscale and can be applied to any type of substrate material. 2-Methyl-2-oxazoline and 2-ethyl-2-oxazoline were used as a precursor for plasma polymerisation. When prepared under appropriate parameters the coatings have excellent biocompatibility and substantially reduce bacterial colonisation. We show that the stability of S. epidermidis biofilms on such surfaces produced under specific parameters was significantly reduced and the biofilms detached during routine rinsing steps. The biocompatibility of the coatings was examined in culture of primary human derived dermal fibroblast cells. The viability of the cells was similar to that when the cells are cultured on surfaces that are known to be favoured by these cells. This combination of excellent biocompatibility and capacity to reduce biofouling offer great opportunities for application in the biomedical fields such as coatings for catheters and various implantable and laboratory devices. The coatings may also find application in the delivery of pharmaceuticals and biosensor technologies. Nonmedical applications where biofouling is a problem may also be an opportunity.

[00133] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

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Claims

1. A plasma polymerised polyoxazoline polymer.

2. The plasma polymerised polyoxazoline polymer of claim 1, wherein the polyoxazoline polymer has been formed by plasma polymerisation of an oxazoline monomer that is a substituted oxazoline with a substituent at any of the 2-, 4- or 5-positions of the oxazoline ring or any combination of these substituents.

3. The plasma polymerised polyoxazoline polymer of claim 2, wherein the oxazoline monomer is selected from the group consisting of 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2,4-disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, and 2,4,5-trisubstituted oxazolines.

4. The plasma polymerised polyoxazoline polymer of claim 3, wherein the oxazoline monomer comprises a 2-substituted-2 -oxazoline.

5. The plasma polymerised polyoxazoline polymer of claim 4, wherein the 2-substituted-2- oxazoline is a 2-alkyl-2-oxazoline.

6. The plasma polymerised polyoxazoline polymer of claim 5, wherein the 2-alkyl-2-oxazoline is 2- methyl -2 -oxazoline.

7. The plasma polymerised polyoxazoline polymer of claim 5, wherein the 2-alkyl-2-oxazoline is 2- ethyl-2-oxazoline.

8. The plasma polymerised polyoxazoline polymer of any one of claims 1 to 7, wherein the polyoxazoline polymer has been fonned by plasma polymerisation under conditions comprising a power of greater than 30W for a time of greater than 5 minutes.

9. The plasma polymerised polyoxazoline polymer of any one of claims 1 to 8, wherein the polyoxazoline polymer comprises intact oxazoline rings.

10. A substrate comprising a plasma polymerised polyoxazoline polymer of any one of claims 1 to 9 on a surface thereof.

11. A substrate comprising an antibiofouling surface, wherein the antibiofouling surface comprises the plasma polymerised polyoxazoline polymer of any one of claims 1 to 9.

12. A substrate comprising a plasma polymerised polyoxazoline polymer film of any one of claims 1 to 9 on a surface thereof and one or more ligands and/or biomolecules bonded to the polyoxazoline polymer film.

13. The substrate of claim 12, wherein the biomolecule is selected from amino acids, peptides, proteins, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents, antithrombogenic agents, and cell attachment proteins.

14. A process for preparing a plasma polymerised polyoxazoline polymer film on a surface of a substrate, the process comprising exposing the surface of the substrate to a plasma comprising an oxazoline monomer vapour under conditions to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer on the surface of the substrate.

15. The process of claim 14, wherein the oxazoline monomer is a substituted oxazoline with a substituent at any of the 2-, 4- or 5-positions of the oxazoline ring or any combination of these substituents.

16. The process of claim 15, wherein the oxazoline monomer is selected from the group consisting of 2-substituted oxazolines, 4-substituted oxazolines, 5-substituted oxazolines, 2,4-disubstituted oxazolines, 2,5-disubstituted oxazolines, 4,5-disubstituted oxazolines, and 2,4,5-trisubstituted oxazolines.

17. The process of claim 16, wherein the oxazoline monomer comprises a 2-substituted-2-oxazoline.

18. The process of claim 17, wherein the 2-substituted-2-oxazoline is a 2-alkyl-2-oxazoline.

19. The process of claim 18, wherein the 2-alkyl-2 -oxazoline is 2-methyl-2-oxazoline.

20. The process of claim 18, wherein the 2-alkyI-2-oxazoline is 2-ethyl-2-oxazoline.

21. The process of any one of claims 14 to 20, wherein the conditions required to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer comprise a power of greater than 30W for a time of greater than 5 minutes.

22. The process of any one of claims 14 to 21 , wherein the conditions required to polymerise the oxazoline monomer to form the plasma polymerised polyoxazoline polymer result in the polyoxazoline polymer comprising intact oxazoline rings.

23. A substrate formed by the process of any one of claims 14 to 22.

24. A process for immobilising a biomolecule on a surface of a substrate, the process comprising providing a substrate comprising a plasma polymerised polyoxazoline polymer according to any one of claims 1 to 9 on a surface thereof, and contacting the plasma polymerised polyoxazoline polymer film with the biomolecule or a derivative or precursor thereof under conditions to bind the biomolecule to the plasma polymerised polyoxazoline polymer film.

25. The process of claim 24, wherein the biomolecule is selected from amino acids, peptides, proteins, aptamers, nucleic acids, DNA molecules, RNA molecules, antibodies, growth factors, antimicrobial agents, antithrombogenic agents, and cell attachment proteins.

26. A substrate formed by the process of any one of claims 24 to 25.