WO2015184492A1 - Bacteriostatic surfaces - Google Patents

Abstract

The present disclosure relates to substrates comprising an antibacterial surface, the antibacterial surface comprising a nitrite containing polymer and to substrates comprising a bacteriostatic surface, the bacteriostatic surface comprising a nitrite containing polymer. The disclosure also relates to processes for preparing a substrate comprising an antibacterial and/or bacteriostatic surface, methods for preventing or inhibiting bacterial growth on a surface of a substrate, and methods for selectively inhibiting or preventing the growth of bacterial cells on a substrate when it is used in the presence of eukaryotic cells.

Description

BACTERIOSTATIC SURFACES

PRIORITY DOCUMENTS

[0001 ] The present application claims priority from Australian Provisional Patent Application No. 2014902149 titled "BACTERIOSTATIC SURFACES" and filed on 5 June 2014, the content of which is hereby incorporated by reference in its entirety.

INCORPORATION BY REFERENCE

[0002 ] The following publications are referred to in the present application and their contents are hereby incorporated by reference in their entirety:

• Witte M.B. and Barbul A, Am J Surg. 2002;183(4):406-12

• Griesser, Vacuum 1989; 39(5): 485-488

• Coad, B.R., et al, ACS Applied Materials & Interfaces, 2012. 4(5): p. 2455-2463

• Al Masum, M, et al. Tetrahedron Lett. 2014; 55( 10): 1726-1728

[0003 ] The content of each of these applications is hereby incorporated by reference in their entirety. TECHNICAL FIELD

[0004] The present disclosure relates to substrates having bacteriostatic surfaces, to methods for making such substrates and surfaces and to uses of the bacteriostatic surfaces to inhibit bacterial growth.

BACKGROUND

[0005 ] The growth of microorganisms, such as bacteria on surfaces needs to be prevented or minimised in a range of settings, such as in some clinical, industrial or domestic applications. For example, medical device related infections account for a substantial morbidity as well as causing a sharp increase in healthcare costs. Drinking water systems are known to harbor bacteria laden surfaces and water cooling towers for air conditioners are well known to pose public health risks from bacterial growth, such as episodic outbreaks of infections like Legionnaires' disease. These are some of the reasons why there is a continual need to develop surfaces of medical devices, impl ants, food packaging, industrial pipes and water treatment and storage that inhibit or prevent bacterial growth.

[0006 ] The accumulation of bacteria on surfaces usually takes place through the formation of biofilms. Biofilms are formed when microorganisms, especially bacteria, attach to surfaces and secrete a hydrated polymeric matrix that surrounds them. Biofilms grow slowly, in one or more locations, colonised by one or a plurality of microorganisms. The pattern of biofilm development involves initial attachment of a microorganism to a solid surface, the formation of colonies attached to the surface, and finally the differentiation of the colonies into mature biofilms. Planktonic cells are released from biofilms and, in this way the biofilm is a source of invasive infections. Many antibacterial treatments treat the infection caused by the planktonic bacteria, but fail to kill bacteria in the biofilm.

[0007] In many settings, biofilm based infections are difficult to eradicate. A promising strategy to prevent bacterial attachment and growth on surfaces is to apply an ultrathin antibacterial coating onto materials and devices. For example, inorganic materials, such as silver or copper ions, or organic materials, such as antibiotics, can be chemically or non-chemically bonded to a surface to form a coating which is either toxic to microorganisms or inhibits the growth of microorganisms on the surface. Despite these advances, contamination of devices and infection therefrom continues to be a problem.

[0008J There is thus a need to provide new or alternative materials and methods of coating substrates with a material which exhibits bacteriostatic properties to reduce the potential for bacterial colonisation on the surface.

SUMMARY

[0009 ] The inventors have found that plasma polymerisation can be used to deposit coatings that release nitric oxide (NO) and achieve antibacterial activity with substantially no adverse effects on mammalian cells. This provides selective antibacterial coatings that can be used in a range of areas, including (but not limited to) medical devices and implants.

[0010] According to a first aspect, there is provided a substrate comprising an antibacterial surface, the antibacterial surface comprising a nitrite containing polymer.

[001 1 ] We have shown that the antibacterial surface inactivates, prevents or inhibits bacterial growth and biofilm formation. Accordingly, in a second aspect there is provided a substrate comprising a

bacteriostatic surface, the bacteriostatic surface comprising a nitrite containing polymer.

[0012] In embodiments of the first and second aspects, the nitrite containing polymer is a plasma polymer. Thus, according to a third aspect there is provided a process for preparing a substrate comprising an antibacterial and/or bacteriostatic surface, the process comprising exposing a substrate and an organic nitrite to a plasma environment under conditions to deposit a nitrite containing plasma polymer on the surface. [0013 J According to a fourth aspect, there is provided a method for preventing or inhibiting bacterial growth on a surface of a substrate, the method comprising forming an antibacterial and/or bacteriostatic surface comprising a nitrite containing polymer on the substrate and exposing the substrate to an environment containing water that is susceptible to bacterial infection.

[0014 ] Advantageously, the antibacterial and/or bacteriostatic surface shows no adverse effect on eukaryotic cells. Accordingly, in a fifth aspect the present invention provides a method for selectively inhibiting or preventing the growth of bacterial cells on a substrate when it is used in the presence of eukaryotic cells, the method comprising forming an antibacterial surface comprising a nitrite containing polymer on the substrate and exposing the substrate to an environment containing water that is susceptible to bacterial infection.

[0015] In embodiments of the fourth and fifth aspects, the nitrite containing polymer is a plasma polymer. Thus, in embodiments of the first to fifth aspects the nitrite containing plasma polymer is prepared by plasma polymerisation of one or more organic nitrites. The organic nitrite may be an alkyl nitrite, aryl nitrite, alkenyl nitrite or alkynyl nitrite.

[0016] In embodiments, the organic nitrite is an alkyl nitrite. The alkyl nitrite may be a Ci -C₂o alkyl nitrite. Examples of suitable Ci-C₂o alkyl nitrites include methyl nitrite, ethyl nitrite, isopropyl nitrite, butyl nitrite, isobutyl nitrite, pentyl nitrite, and isopentyl nitrite. In specific embodiments, the alkyl nitrite is isopentyl nitrite (IPN).

[0017] In other embodiments, the organic nitrite is an alkenyl nitrite. The alkenyl nitrite may be a C₃-C₁₂ alkene which can be linear or branched and in which a hydrogen atom has been replaced by a nitrite (- ONO) group. Examples of suitable alkenyl nitrites include isoprenyl nitrite (ie. 2-methylbutenyl nitrite), allyl nitrite, butenyl nitrite, and pentenyl nitrite. In specific embodiments, the alkenyl nitrite is isoprenyl nitrite.

[0018] In still further embodiments, the organic nitrite is an alkynyl nitrite. The alkynyl nitrite may be a C3-C20 alkyne which can be linear or branched and in which a hydrogen atom has been replaced by a nitrite (-ONO) group. Suitable alkynyl nitrites include, for example, propyne nitrite, butyne nitrite, etc.

[0019] In still other embodiments, the organic nitrite is an aryl nitrite. Suitable aryl nitrites include, for example, phenyl nitrite, p-tolyl nitrite, p-methoxyphenyl nitrite, etc.

[0020] The substrate may be a metal, synthetic polymer, biopolymer, glass or ceramic material or a combination of any of these materials. Suitable synthetic polymers (plastics) include, but are not limited to polytetrafluoroethylene (Teflon), polyethylene, polypropylene (PP), polydimethylsiloxane (PDMS), polystyrene (PS), poly( ether sulfone), polyacrylonitrile, cellulose acetate, polyvinylidene fluoride (PVDF), polysulfone, polyamide, polyurethane, poly(tetrafluoroethylene-co-hexafluoropropylene) (FEP), poly( ethylene tereplithalate) (PET), poly(4-methyl- 1 -pentene) (PMP), and polyether ether ketone (PEEK). Suitable biopolymers include, but are not limited to hydroxyapatite, collagen, and elastin. Suitable metals include, but are not limited to copper, iron, aluminium, titanium, stainless steel, and steel. Suitable ceramics include, but are not limited to SiC/Al₂0₃ ceramic and AI₂O₃/S1O₂ ceramic.

[0021 ] The substrate may be any device that comes into contact with an environment that is susceptible to bacterial infection. For example, the substrate may be a medical device, an implant, a water treatment device or part thereof, a food packaging container, a food handling device or part thereof, an industrial pipe, a water tower, and the like.

BRIEF DESCRIPTION OF DRAWINGS

[0022] Embodiments of the present invention will be discussed with reference to the accompanying drawings wherein:

[00231 Figure 1 shows XPS data for high-resolution C Is, N I s and O Is;

[0024 ] Figure 2 is a plot of N0₂ ^" concentration following hydrolysis of coatings in either phosphate buffered saline (PBS) or in a pH 13 adjusted aqueous solution at either 37 °C or 70 °C over the course of 72 h;

[0025 ] Figure 3 shows BacLight time lapse images of a reference substrate (air-plasma treated PET "REF") and IPNpp coated coverslips incubated with Staphylococcus epidermidis in tryptic soy broth after 0, 9 and 14 hours;

[0026] Figure 4 is a Live/Dead image of a reference substrate (air-plasma treated PET) incubated with Staphylococcus epidermis in tryptic soy broth after 0, 1 1 and 15 hours;

[0027] Figure 5 is a Live/Dead image of IPN plasma polymer coated coverslips incubated with

Staphylococcus epidermis in tryptic soy broth after 0, 11 and 15 hours;

[0028] Figure 6 shows images of MSC stained with DAP1 (nuclei, blue) and phalloidin (actin filaments, red); and

[ 0029] Figure 7 shows images of stained bacterial biofilms, grown in wells of a 24-well plate, on different surface chemistries after the indicated periods of time of growth. Column (a) is an uncoated control polystyrene surface; (b) is an isoprenyl nitrite plasma polymer coated surface; (c) is an isopentyl nitrite plasma polymer coated surface; (d) is an uncoated control polystyrene surface; (e) is an isoprenyl nitrite plasma polymer coated surface; and (f) is an isopentyl nitrite plasma polymer coated surface.

DESCRIPTION OF EMBODIMENTS

[0030J Provided herein is a substrate comprising an antibacterial surface. The antibacterial surface comprises a nitrite containing polymer coating on a bulk substrate material or device.

[0031 ] The nitrite containing polymer is preferably a plasma polymer. For the ease of discussion, reference will now be made to a nitrite containing plasma polymer. However, it will be understood that the invention is not restricted to such embodiments and it is contemplated that the nitrite containing polymer could also be formed by conventional polymerisation of monomer(s) that contain nitrite groups using standard polymerisation conditions.

[0032] Advantageously, the nitrite containing plasma polymers are stable at room temperature and under dry conditions over a period of several months. However, upon contact with water or other aqueous fluids they release NO. NO is an important signalling molecule in the quorum sensing of bacteria, which regulates bacterial proliferation and biofilm formation. The NO released from the nitrite containing plasma polymer when the surface comes in to contact with water causes a halt in the growth of bacteria and biofilm formation. This effect has been observ ed for up to 12h. Advantageously, our data also show that there is no adverse effect on eukaryotic cells. Accordingly, the present invention also provides a method for selectively inhibiting or preventing the growth of bacterial cells on a substrate when it is used in the presence of eukaryotic cells, the method comprising forming an antibacterial surface comprising a nitrite containing plasma polymer on the substrate and exposing the substrate to an environment containing water that is susceptible to bacterial biofilm formation.

[0033] The nitrite containing plasma polymer can be deposited onto any suitable substrate such as a hard or a soft substrate. The substrate may be a metal, synthetic polymer, biopolymer, glass or ceramic material or a combination of any of these materials. Suitable synthetic polymers (plastics) include, but are not limited to polytetrafluoroethylene (Teflon), polyethylene, polypropylene (PP), polydimethylsiloxane (PDMS), polystyrene (PS), poly( ether sulfone), polyacrylonitrile, cellulose acetate, polyvinylidene fluoride (PVDF), polysulfone, polyamide, polyurethane, poly(tetrafluoroethylene-co- hexafluoropropylene) (FEP), poly(ethylene terephthalate) (PET), poly(4-methyl-l-pentene) (PMP), and polyether ether ketone (PEEK). Suitable biopolymers include, but are not limited to hydroxyapatite, collagen, and elastin. Suitable metals include, but are not limited to copper, iron, aluminium, titanium, stainless steel, and steel. Suitable ceramics include, but are not limited to SiC/Al₂0₃ ceramic and Al₂0₃/Si0₂ ceramic. The substrate may be a composite material comprising any suitable material coated with any one or more of the aforementioned materials. [0034] The substrate may be any device that comes into contact with an environment containing water that is susceptible to bacterial infection. For example, the substrate may be a medical device, an implant, a water treatment device or part thereof, a food packaging container, a food handling device or part thereof, an industrial pipe, a water tower, and the like.

[00351 In embodiments, the substrate is a medical device. The medical device may be an implantable or non-implantable device, such as a replacement joint, a urinary catheter, a percutaneous access catheter, an endotracheal tube, a stent, a pacemaker, a prosthetic, a bandage, a wound dressing, or a contact lens. The medical device may be formed from stainless steel, titanium, polypropylene titanium, hydroxyapatite, polyethylene, polyurethanes, organosiloxane polymers, perfluorinated polymers, acrylic hydrogel polymers, siloxane hydrogel polymers, fibrous bandage and dressing materials, synthetic dressings or hydrogel or foam dressings.

[ 0036] The substrate may also be used for a range of non-medical applications where inhibition of microbial attachment colonisation and/or biofilm formation is desired. For example, industrial surfaces that frequently come into contact with aqueous streams are particularly susceptible to biofilm formation. Examples of non-medical applications include the coating of water treatment equipment, the coating of cooling tower components, the coating of processing equipment particularly in food and pharmaceutical production processes, and the coating of packaging for foods and pharmaceuticals. Non-medical devices of this type may be formed from materials such as polypropylene, polystyrene, polyethylene

terephthalate, polyester, polyamides, polyvinyl chloride, polyurethanes, polycarbonates, polyvinylidene chlorides, polyethylene, stainless steel, steel, iron or tin.

[0037] In embodiments, the nitrite containing plasma polymer is prepared by plasma polymerisation of one or more organic nitrites. Thus, provided herein is a process for preparing a substrate comprising an antibacterial surface, the process comprising exposing a substrate and an organic nitrite to a plasma environment under conditions to deposit a nitrite containing plasma polymer on the surface. Optionally a carrier gas such as argon may be added to assist in forming a stable plasma glow discharge, as is well known to those skilled in the art.

[0038] As used herein, the term "organic nitrite" means a carbon-based molecule that has at least one nitrite moiety as part of the molecule. The carbon skeleton of the organic nitrite may comprise 1 to 20 carbon atoms (ie. Ci-C₂o), and may be saturated or unsaturated. Suitable organic nitrites include alkyl nitrites, aryl nitrites, alkenyl nitrites and alkynyl nitrites.

[0039] Alkyl nitrites are chemical compounds having the molecular structure RONO where R is an alkyl group. Alkyl nitrites are alkyl esters of nitrous acid (ΗΟΝΌ). The alkyl nitrite may be a Ci-C₂o alkyl nitrite, such as a Q alkyl nitrite, a C₂ alkyl nitrite, a C₃ alkyl nitrite, a C₄ alkyl nitrite, a C₅ alkyl nitrite, a C₆ alkyl nitrite, a C₇ alkyl nitrite, a C alkyl nitrite, a C₉ alkyl nitrite, a C_l0 alkyl nitrite, a Cn alkyl nitrite, a C]₂ alkyl nitrite, a C₎₃ alkyl nitrite, a C_w alkyl nitrite, a Cj₅ alkyl nitrite, a Cu, alkyl nitrite, a Cn alkyl nitrite, a C_{] 8} alkyl nitrite, a Ci₉ alkyl nitrite or a C₂o alkyl nitrite. Suitable alkyl nitrites include, for example, methyl nitrite, ethyl nitrite, isopropyl nitrite, butyl nitrite, isobutyl nitrite, pentyl nitrite, isopentyl nitrite, etc. In specific embodiments, the alkyl nitrite is isopentyl nitrite. Suitable alkyl nitrites may be commercially available or can be prepared using the method described in W. A. Noyes, Org. Synth. 1936, 16, 7.

[0040] The organic nitrite may be an alkenyl nitrite. The alkenyl nitrite may be a C₃-Ci₂ alkenyl nitrite, such as a C] alkenyl nitrite, a C₂ alkenyl nitrite, a C₃ alkenyl nitrite, a C₄ alkenyl nitrite, a C₅ alkenyl nitrite, a C₆ alkenyl nitrite, a C₇ alkenyl nitrite, a C₈ alkenyl nitrite, a C₉ alkenyl nitrite, a C_]0 alkenyl nitrite, a Cn alkenyl nitrite or a Cn alkenyl nitrite. Suitable alkenyl nitrites include, for example, vinyl nitrite, allyl nitrite, butenyl nitrite, pentenyl nitrite, isoprenyl nitrite (ie. 2-methylbutenyl nitrite), but-2- enyl nitrite, 2-methylpropenyl nitrite, pent-2-enyl nitrite, 2-methylbut-2-enyl nitrite, 3-methylbut-2-enyl nitrite, etc. Suitable alkenyl nitrites may be commercially available or can be prepared using the method described in W. A. Noyes, Org. Synth. 1936, 16, 7.

[0041 ] The organic nitrite could be an alkynyl nitrite. The alkynyl nitrite may be a C₃-C_{] 2} alkyne which can be linear or branched and in which a hydrogen atom has been replaced by a nitrite (-ONO) group. The alkynyl nitrite may be a C₃-Ci₂ alkynyl nitrite, such as a Cj alkynyl nitrite, a C₂ alkynyl nitrite, a C₃ alkynyl nitrite, a C₄ alkynyl nitrite, a C₅ alkynyl nitrite, a C₆ alkynyl nitrite, a C₇ alkynyl nitrite, a C₈ alkynyl nitrite, a C₉ alkynyl nitrite, a Cm alkynyl nitrite, a Cn alkynyl nitrite or a Cn alkynyl nitrite. Suitable alkynyl nitrites include, for example, propyne nitrite, butyne nitrite, etc. Suitable alkynyl nitrites may be commercially available or can be prepared using the method described in W. A. Noyes, Org. Synth. 1936, 16, 7.

10042] The organic nitrite could be an aryl nitrite. Aryl nitrites may be less advantageous than alkyl nitrites due to their lower volatility. The aryl nitrite may be any relatively volatile compound that contains a nitrite group and an aromatic ring. Examples of aromatic rings include benzene, naphthalene, pentalene, indene, azulene, heptalene, biphenylene, indacene, acenaphthylene, fluorene, phenalene, anthracene, fluoranthene, acephenanthrylene, aceanthrylene, triphenylene, biphenyl, pyrene, chrysene, naphthacene, pleiadene, picene, perylene, pentaphene, pentacene, tetraphenylene, hexaphene, hexacene, rubicene, coronene, trinaphthylene, heptaphene, heptacene, pyranthrene, ovalene, indan, tetralin, acenaphthene, cholanthrene, aceanthrene, acephenanthrene, violanthrene, isoviolanthrene, naphthopezylene, indenoindene, benzocyclooctene and the like. The aryl nitrite could contain a heterocyclic ring, such as a furan, thiophene, pyrrole, isopyrrole, pyrazole, imidazole, isoimidazole, triazole, dithiole, oxathiole, isoxazole, oxazole, thiazole, isothiazole, oxadiazole, oxatriazole, dioxazole, oxathiazole, oxathiole, pyran, pyrone, dioxin, pyridine, pyridizine, pyrimidine, pyrazine, triazine, oxazine, isoxazine, oxathiazine, oxadiazine, azepine, benzazepine, oxepin, benzoxepin, thiepin, benzthiepin, diazepin, benzdiazepin, benzofuran, isobenzofuran, thionaphthene, isothionaphthene, indole, isoindole, indolenine, pyrindine, pyranopyrazole, benzpyrazole, benzisoxazole, benzoxazole, anthranil, benzopyran, benzopyrone, quinoline, isoquinoline, cinnoline, quinazoline, naphthyridine, pyridopyridine, benzoxazine,

benzisoxazine, carbazole, xanthene, acridine, purine, and the like. Suitable aryl nitrites include, for example, phenyl nitrite, p-tolyl nitrite, p-methoxyphenyl nitrite, etc. The aryl nitrites may be

commercially available or can be prepared using the methods described in W. A. Noyes, Org.

Synth. 1936, 16, 7 or Al Masum, M, et al. Tetrahedron Lett. 2014; 55( 10): 1726-1728.

[0043] The organic nitrite may be a mixture of any of the aforementioned organic nitrites.

[0044 ] The "plasma environment" is formed using a plasma source to generate a low temperature gas glow discharge that provides energy to activate the organic nitrite in the gas phase in order to

initiate polymerisation. The plasma environment consists of a mixture of electrons, ions, radicals, neutrals and photons. The plasma apparatus used to generate the plasma environment may be one of those known in the art and polymerisation of the organic nitrite can be carried out at atmospheric pressure in a sealed chamber or at reduced pressure in a vacuum chamber. Thus, the step of exposing the surface of the substrate and the organic nitrite to a plasma environment may include placing the substrate in a chamber, sealing the chamber, introducing a vapour containing the organic nitrite into the chamber, and maintaining the substrate at a temperature suitable for polymerisation of the organic nitrite so as to form a polymer film on the surface. The plasma environment may be generated using electrodes or an RF coil. A suitable plasma apparatus is described by Griesser (Griesser, Vacuum 39(5): 485-488 ( 1989)).

[0045 ] Preferably, the plasma deposition step comprises exposing the substrate and the organic nitrite to low power glow plasma discharge under continuous power and then exposing the substrate and the organic nitrite to pulsed low power glow plasma discharge. This provides a selective and industrially applicable antibacterial coating. In embodiments, the organic nitrite is plasma deposited in a continuous deposition step for a period of from about 1 to about 15 minutes and then in a pulsed deposition step comprising a pulsed deposition rate of from about 1/10 ms to about 1/30 ms for a total period of from about 10 to about 120 minutes. For example, isopentyl nitrite can be plasma deposited using a power of 15 W and pressure of 200mTorr for 2 minutes continuous and then 90 minutes pulsed at a rate of 1/20 ms. This creates a surface which is bacteriostatic for at least 12 hours. These plasma conditions and durations are specific to the laboratory equipment used by the inventors; it is well known in the art that industrial- scale plasma systems can be designed to be more efficient, thus enabling shorter plasma processing times.

[0046] Optionally, the surface of the substrate may be treated prior to deposition of the nitrite containing plasma 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, nitrogen or argon in a plasma chamber in order to activate and/or clean the surface.

[0047] The plasma polymerisation conditions may be used to control the thickness of the coating and the thickness of the coating may correlate with the amount of NO released when the nitrite containing polymer comes in to contact with water. However, the nitrite containing plasma polymer preferably forms a thin film coating. The thickness of the coating may be less than about 4μιη, and is preferably from about 5 nm to about 250 nm in thickness.

[0048 ] The plasma polymerisation conditions can also be used to control the density of crosslinks in the nitrite containing plasma polymer and this may be used to control the rate of permeation of water into the nitrite containing polymer and, therefore, the rate of release of NO from the polymer.

[0049] The nitrite containing plasma polymer may also be part of a multi-layered coating comprising two or more polymer film layers. The coating may comprise first and second polymer film layers. The first film layer may be the layer in contact with the surface of the substrate and may comprise the nitrite containing plasma polymer. The second film layer may, for example, may be an overcoat layer that modifies the rate of permeation of water into the first film layer so as to modify the rate of formation and release of NO. The second film layer may also be a functional layer and may, for example, comprise growth factors to assist in host cell attachment and/or growth, or polyethers such as poly(ethylene oxide) to confer resistance to biological adhesion.

[0050] Ideally, the plasma deposition process provides coatings that are uniform, strongly adhered to the surface of the substrate, selective in their action, easy to produce with efficient deposition and high process reproducibility, and can be produced at a relatively low unit cost.

[0051 ] The antibacterial surface inactivates, prevents, or inhibits bacterial growth and biofilm formation. Our results suggest biomolecular interference in that the surfaces do not kill bacteria, but the bacteria do not agglomerate into a biofilm and do not stick to the surface. Thus, the surface may be referred to as a "bacteriostatic" surface. The invention therefore also provides a method for preventing or inhibiting bacterial growth on a surface of a substrate, the method comprising forming an antibacterial surface comprising a nitrite containing plasma polymer on the substrate and exposing the substrate to an aqueous environment that is susceptible to bacterial infection.

[0052] Antibacterial surfaces formed according to the methods described herein can be stored at room temperature without significant loss of antibacterial activity. Specifically, a substrate comprising an antibacterial surface as described herein was allowed to stand at room temperature for a month and the antibacterial activity of the surface was then tested against Staphylococcus epidermidis and the results were compared to substrates that had been refrigerated for the same period. The storage conditions appeared to make little to no difference in activity compared with fresh samples.

[0053 ] Advantageously, the nitrite containing polymer coatings show no adverse effects on eukaryotic cells. As such, also provided herein is a method for selectively inhibiting or preventing the growth of bacterial cells on a substrate when it is used in the presence of eukaryotic cells, the method comprising forming an antibacterial surface comprising a nitrite containing plasma polymer on the substrate and exposing the substrate to an environment containing water that is susceptible to bacterial infection.

[0054 ] The antibacterial surface described herein may be particularly suitable for use on wound dressings or bandages because NO assists in wound healing (Witte M.B. and Barbul A, Am J

Surg. 2()02; 183(4):406- 12). Wound healing is a complex, sequential cascade of events and NO formed from L-arginine regulates collagen formation, cell proliferation and wound contraction. Thus, wound dressings or bandages comprising the antibacterial surface described herein may assist in wound healing by releasing NO upon contact with moisture in the wound in addition to preventing the growth of bacteria thereon.

[0055] The invention is hereinafter further described by way of the following non-limiting examples and accompanying figures.

EXAMPLES

[0056] Example 1 - Plasma polymerised isopentyl nitrite polymers (IPNpp) [0057] Materials

[0058] Isopentyl nitrite 96% (IPN) was purchased from Sigma-Aldrich (St. Louis, MO) and used as received. The coverslips were stamped out with a 13 mm biopsy punch from a 0.05 mm thick PET foil supplied from Goodfellow Cambridge Ltd. (Huntingdon, England) and washed with ethanol twice prior to use. Oxoid™ Nutrient Agar (CM0003) and cold filterable Oxoid™ Tryptic Soy Broth (TSB, CM 1065) were purchased from Thermofisher (Scoresby, Australia). Phosphate buffered saline (PBS, SLBB6584) tablets and Safranin stain were purchased from Sigma Aldrich. The Bac Light™ staining kit was purchased from Invitrogen (Mulgrave, Australia) and used according to specifications. All chemicals were used as received and Milli-Q™ filtered water was used to prepare solutions, according to recommended concentration. For antibacterial testing, the bacterial strain used was Staphylococcus epidermidis ATCC® 35984™. 24 well plates were NUNC™ brand, purchased from Thermofisher.

[0059] Plasma polymerization [0060 ] Plasma polymerization (Griesser, H.J., Small scale reactor for plasma processing of moving substrate web. Vacuum, 1989. 39(5): p. 485-488 and Coad, B.R., et al., Functionality of Proteins Bound to Plasma Polymer Surfaces. ACS Applied Materials & Interfaces, 2012. 4(5): p. 2455-2463) was carried out as follows: PET coverslips and one silicon wafer were placed into the plasma chamber. The silicon wafer was used for determination of the IPN plasma polymer coating thickness by ellipsometry. First, the plasma chamber was pumped down to a base pressure of 30 mTorr. Then the air inlet valve was carefully opened to allow for air flowing into the chamber so as to stabilize the pressure at 200 mTorr. An air plasma with a RF frequency of 13.56 MHz, at a vapour pressure of 200 mTorr, input power of 50 W and treatment time of 1 minute was used to oxidize the surface of the PET coverslips in order to ensure better bonding of the subsequently deposited pl asma polymer coating; fol lowed by evacuation of the plasma chamber again to a base pressure of 30 mTorr. IPN vapour was introduced into the plasma chamber and the flow adjusted so that the pressure was stabilised at 200 mTorr. Then plasma polymerization was performed with a RF frequency of 13.56 MHz, at a vapour pressure of 200 mTorr, input power of 18 W and deposition time of 2 minutes under continuous plasma irradiation. Afterwards the plasma power input method was switched to pulsed mode ( 1 ms on / 20 ms off time) for a period of 90 minutes while the pressure was maintained at 200 mTorr. The deposition conditions are summarized in Table 1.

Table 1 - Plasma polymerisation conditions

[0062] The atomic composition and thickness of the resulting plasma polymer, as determined by XPS and ellipsometry, are summarised in Table 2.

[0063 ] The surface analysis was carried out using a Kratos Axis Ultra DLD spectrometer, utilizing a monochromatic Al Ka X-ray source running at 225W, corresponding to an energy of 1486.6 eV. The area of analysis was 0.3*0.7 mm and an internal flood gun was used to supress the charging of the samples. Survey spectra were collected at 160 eV pass energy with steps of 0.5 eV and a dwell time of 55 ms. High resolution spectra were collected at 20 eV pass energy an 0.1 eV steps for O 1 s, N I s and C 1 s. The data was processed and analysed with CasaXPS (ver.2.3. 16 Casa Software Ltd.) utilizing Shirley baseline correction. To compensate for charging effects, all spectra were offset the C I peak corresponded to 284.8 eV. Atomic percentages were rounded to one decimal after the coma. [0064] The thickness of the deposited IPN plasma polymer was determined using a J.A. Woollam (Model MC-200) V-Vase ellipsometer. For this purpose IPN was deposited onto a silicon wafer under standard conditions, as described above; followed by ellipsometry measurement over a wavelength range of 400-1 100 nm in 10 nm steps at alignment angles of 65°, 70° and 75°. The experimental data was fitted using the supplied modelling software WVASE32 (Ver. 3.770), utilizing a two layer Cauchy model. By refining the optical parameters, the mean squared error of the fi t was minimized and the plasma polymer thickness was obtained.

[0065] Table 2 - XPS and ellipsometry data

[0066 ] The obtained atomic percentages correspond well with the theoretical values of a sufficiently thick IPN coating which would amount to 67.5% C, 12.5% N and 25% O. The slightly lower percentage of nitrogen and oxygen is an indicator of a low degree of fragmentation and elimination of the labile nitrosoxy functional group.

[0067] Curve fitting of high-resolution C Is, N Is (Figure I ) and O Is show the majority of N species being found in C-N bonds, which is possible after the fragmentation of the nitrosoxy group (0-N-O) and recombination with C species. However, all spectra support the presence of the nitrosoxy group at a non- negligible level. Hand in hand, the N Is as well as O Is spectra suggest that 9% of nitrogen and 24% of oxygen were bound in form of the desired nitrosoxy group; pointing towards an overall retention of roughly one tenth of the functional group stemming from the IPN precursor.

[0068] To quantify the release of the short lived NO, it is necessary to capture the molecule in the more stable oxidized form of N02^~ and quantify through the colorimetric Griess assay. NO in coatings was hydrolysed in either phosphate buffered saline (PBS) or in a pH 13 adjusted aqueous solution at either 37 °C or 70 °C over the course of 72 h (Figure 2).

[0069] It is evident that the amount of NO released, which directly corresponds to the NO2^" measured, depended upon the immersion media and temperature. A temperature increase in either case led to an almost three times higher amount of released NO; the same roughly applied when the pH of the solution was increased. The elevated release of NO may be used to advantage in post-operational infection, where it is known that there is an increase of temperature and pH in the wound environment, thus leading to an increase in NO release when it is required.

[0070 ] Bacterial testing - Methods

[0071 ] 1 mL of a frozen aliquot (-20 °C) of bacteria were thawed, followed by plating a small portion out on an agar plate and incubated overnight at 37 °C. The frozen 1 mL aliquots were replaced new ones from the -78 °C freezer once a month in order to ensure the genetic pedigree of the bacteria. The next day a single colony was picked and seeded into 10 mL of TSB and incubated at 37 °C for at least 18 h. 10 mL of this solution were centrifuged at 1 OOOrpm for 10 minutes, removing the supernatant and replacing it with PBS; followed by two subsequent centrifugation/wash cycles with PBS. The optical density of the bacteria in PBS solution was determined at 600 nm and adjusted with fresh PBS so its optical density corresponded to OD600=0.2 which approximately corresponds to a bacterial concentration of 10^s CFU/mL. This solution was further diluted with PBS to 10⁶ CFU/mL. The IPN plasma polymer coated and air plasma only treated PET coverslips as reference were placed in a 24-well plate and inoculated with 600 μΐ of the bacteria solution. The well plate was rhythmically shaken at a frequency of 2 Hz and incubated for lh at a temperature of 37 °C to ensure the homogenous attachment of bacteria to the surfaces. Afterwards, the bacteria solution was drawn off and the samples were washed twice with 600 μΐ of PBS; followed by the addition of 600 μΐ of TSB into each well. For clarity sake, this time point was defined as "0 h" and the samples were incubated further under agitation. The samples for analysis were removed from incubation at set time interv als and placed into a separate 24 well plate and innoculated with 300 μΐ of BacLight™ solution which was previously prepared according to specification. After 20 minutes of incubation at room temperature, the samples were washed three times with an excess of deionized water and stored in 600 μΐ of deionized water to prevent the stain drying out, which would cause an alteration of the results. One at a time, the samples were imagined with a Nikon Eclipse Ni™ microscope, equipped with a green/red filter and with the Nikon digital sight DS-L3™ at a 490 nm excitation wavelength. Imagined samples were refrigerated at 4°C to stop any growth. This procedure was repeated for all samples after the specified time interval. Upon all samples were imagined, the supernatant was removed, replaced with 600 μΐ of crystal violet stain and incubated for 20 minutes. This was followed by a washing with an excess of deionized water and placement into a fresh wellplate. Pictures were taken using a Samsung Galaxy S2.

[0072] Bacterial testing - Results

[0073 ] The in vitro testing was performed using a particularly vigorous biofilm forming and clinically relevant strain of Staphylococcus epidermidis (ATC035984™). BacLight staining was employed for qualitative evaluation of the proliferation shown in time lapse results (Figures 3-5). Upon seeding, bacteria densities did not markedly differ between the reference and IPN polymer coated surface (IPNpp); few dead bacteria could be identified at this time point. After incubation, bacteria in the reference sample continued multiplying and formed biofilms. In contrast, the bacteria in contact with the IPNpp neither multiplied nor formed any visible biofilm; this effect persisted up to 14 hours. These qualitative measurements accorded with a quantitative assessment of the total biomass with the help by crystal violet staining of the samples. Even by the naked eye a striking difference could be seen at the 14 hour mark between the reference and the IPNpp coated sample. The test was repeated at different time-points and yielded similar results in all cases; namely the markedly delayed onset of bacterial multiplication and biofilm formation by at least 8 hours or more in all cases.

10074] While organic nitrites can deteriorate over time, presenting a challenge for industrial

manufacturing, we have found that this did not pose a problem for the IPN plasma coatings, as storing samples for 2 months at room temperature and ambient atmosphere prior to use led to little to no difference in performance.

[0075 ] Cytotoxic testing

[ 0076] To investigate biocompatibility of the IPN coatings, cytotoxicity testing with human

mesenchymal stem/stromal cells (MSC) was conducted. A known cytotoxic coating (chlorinated plasma polymer from 1 , 1 , 1 -trichloroethane (TCE)), was used as the negative control.

[0077] Cell Culture

[0078] Human bone marrow aspirate were obtained with written consequent and ethical approval. Every 5 ml bone marrow aspirate was diluted to 35 ml with Phosphate buffer saline (PBS, Invitrogen). The mononuclear cells are isolated using Ficoll-Paque (GE Healthcare) density gradient at 40()g for 20 minutes. The buffy layer was removed and washed in PBS and resuspended in low glucose DMEM (Invitrogen) supplemented with 10 % fetal bovine serum (Thermofisher) and 100 units/mL penicillin, and 100 μg/mL streptomycin (Life Technologies) referred to DMEM 10 % FBS from hereon and seeded into a single T175 flask (Nunc). The cells were then incubated in 20 % oxygen, 5 % C0₂ at 37 °C for 24 hours to allow adherent cells to attach. The media was then replaced with fresh media and the flask was placed into a hypoxic incubator (2 % oxygen and 5% C02 at 37 °C). The media was replaced twice a week until the monolayer was 80-90 % confluent before passaging.

[0079] Kg la myeloid leukaemia cell line was sourced from ATCC and was maintained in RPMI (Invitrogen) supplemented with 10% fetal bovine serum and 100 units/mL penicillin, and 100 μg/mL streptomycin (referred to RPMI 10% FBS from here on) in a T25 flask and incubated at standard cell culture conditions (5% C02 at 37 °C). The media was changed twice weekly and the cells were passage when reach 80-90% confluent.

[0080J MSC cy toxicity assay

[0081 ] Passage 4 MSCs were seeded at 2000 cells/cm² suspended in DM EM 10 % FBS in a 24 well plate that had the surface modified (n=4). For the controls the 2000 cell/cm² are seeded into a normal tissue culture treated 24 well plate suspended in DMEM 10 % FBS for the positive growth control (n=4) and serum free DMEM for the negative control (n=4). For a the non-contact controls, 2000 cells/cm² were seeded in DMEM 10 % FBS into normal tissue culture plate but with surface modified insert place vertically into the well (n=4) and 2000 cell/cm² were seeded in conditioned DMEM 10 % FBS that has been in contact with the modified surface for 24 hours in an incubator (5 % C0₂ at 37 °C) (n=4). The cell was allowed to grow for 4 days in standard tissue culture conditions (37 °C and 5 % C0₂). The cells were quantified by the metabolic probe AlamarBlue (Invitrogen). The media was replaced with a 1 :50 dilution of AlamarBlue in DMEM 10 % FBS and incubated for 3 hours. The fluorescence signal of the

AlamarBlue in media was measured in a plate reader (FLUOstar Omega, BMG Labtech, Germany) along with a cell titration to infer cell numbers using excitation and emission filters of 544 and 590.

[0082] KGIa cy toxicity assay

[0083 ] Kg la were seeded at 20,000 cell/ml suspended in serum free media X-VIVO 15 (Lonza) in a 24 well plate that had the surface modified (n=4). For the controls the 20,000 cell/ml are seeded into a normal tissue culture treated 24 well plate suspended in X-VIVO 15 for the positive growth control (n=4) and serum free RPMI for the negative control (n=4). For a the non-contact controls, 20,000 cells/ml were seeded in X-VIVO 15 into normal tissue culture plate but with surface modified insert place vertically into the well (n=4) and were seeded in conditioned X-VIVO 15 that has been in contact with the modified surface for 24 hours (n=4). The cells were allowed to grow for 4 days in standard tissue culture conditions (37°C and 5% C0₂). The cells were counted by flow cytometry (FC500, Beckman Coulter, USA) using Flow-CountTM Fluorospheres (Beckman Coulter) with 3 μΜ Propidium Iodide to gate out the dead cells.

[0084 ] Cytoxicity Standards

[0085 ] For the MSC 2000 cells/cm² suspended in DMEM 10% FBS in a 24 well plate was titrated with PBS and ethanol to give a dilution and toxicity curve. For the KG l a, the cells were seeded at 20,000 cells/ml suspended in X-VIVO or RPMI 10% FBS was titrated with PBS and ethanol. The cells were allowed to grow for 4 days and the cell number quantified by the AlmarBlue method for the MSC and by flow cytometry for the KG 1 a. [0086] Cell imaging

[0087] MSC monolayers were fixed by submerging in 4% paraformaldehyde ( Sigma- Aldrich) for 20 minutes at RT. Cells were washed twice with PBS, and then actin stained (red) with Alexo Fluor 594 phalloidin (Life Technologies) and nuclei stained with DAPI (Life Technologies) as per the

manufacturers instructions. Stained cells were imaged using an ECLIPSE Ti epifluorescent microscope (Nikon, Japan) coupled to a Nikon DS-Qi l Mc camera.

[0088] The observed cell attachment and spreading was somewhat lower on IPNpp compared with the positive control tissue culture polystyrene (TCP), whereas no viable cells were observed on the cytotoxic control. While the IPNpp coating is less supportive of cell attachment than TCP, this might not matter in practical applications; the key point is that the IPNpp surface has definitely no cytotoxic effect and enables healthy cell proliferation (Figure 6).

[0089] Thus, it has been have shown that suitable plasma conditions can be derived to enable plasma deposition of IPNpp coatings without excessive cleavage of the relatively labile nitrosoxy group in the plasma, and that the resultant coatings demonstrate excellent bacteriostatic properties but no toxicity to stem cells.

[0090] Example 2 - Plasma polymerised isoprenyl nitrite polymers

[0091 1 Following the methods described in Example 1 , PET coverslips were also coated with a coating formed by plasma polymerising isoprenyl nitrite. Isoprenyl nitrite was formed by the following reaction and using the procedure described in W. A. Noyes, Org. Synth. 1936, 16, 7.

[0092] The plasma polymerisation conditions are shown in Table 3. [0093 ] Table 3 - Plasma polymerisation condi tions

Organic Power Pressure Time Time pulsed

Nitrite contiiious

(l/20ms) Isoprenyl 19 W 200 mTorr 2 min 90 min

nitrite

IPN 19 W 200 mTorr 2 min 90 min

[0094 ] The thickenss of the coating fonned from plasma polymerisation of isoprenyl nitrite was measured at 127.477±0. 1 12 nm. I PNpp coatings formed had thicknesses that were measured at 42.522±0.495 nm, 53.936±0.689 nm and 73.933±0.773 nm. These results indicate that the use of an unsaturated monomer yields thicker coatings at the optimal plasma polymerisation conditions.

[0095 1 The results of XPS analysis of the products are shown in Table 4.

[0096] Table 4 - XPS analysis

[00971 Bacterial testing was conducted as described in Examle 1 and the results are shown in Figure 7. The results show that IPN and isoprenyl nitrite plasma polymers performed similarly over the same time period.

[0098] Example 3 - Influence of pressure and power on formation of IPNpp

[0099] Samples were formed using the method described in Example 1 but with the variations in pressure and/or power shown in Table 5.

[00100 ] Table 5 - Varying plasma polymerisation conditions Power (W) Pressure Time CW Time pulsed

(mTorr) (min) (min)

15 100 2 90

35 100 2 90

15 200 2 90

35 200 2 90

[00101 ] We found that pulsing the plasma power input provides well formed coatings. A monomer pressure of 200mTorr, a power of 15W give good results.

1001021 Example 4 - Overcoating oflPNpp

[00103] Samples were formed using the method described in Example 1 and then overcoated with another plasma polymer, as shown in Table 6.

Table 6 - Overcoat deposition conditions

[00105] Bacterial testing as described previously showed that bacteria attach more poorly to hydrophobic overcoats (ie. 1 ,7-octadiene) and that hydrophobic or hydrophilic coatings both reduce overall performance. [00106] 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.

[00107] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00108] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers. The following claims are not intended to limit the scope of the invention.

Claims

1. A substrate comprising an antibacterial surface, the antibacterial surface comprising a nitrite containing polymer.

2. The substrate according to claim 1 , wherein the nitrite containing polymer is a nitrite containing plasma polymer.

3. The substrate according to claim 2, wherein the nitrite containing plasma polymer is prepared by plasma polymerisation of one or more organic nitrites.

4. The substrate according to claim 3, wherein the organic nitrite is a Ci-C₂o organic nitrite.

5. The substrate according to claim 4, wherein the organic nitrite is a Ci-C₂o alkyl nitrite.

6. The substrate according to claim 5, wherein the Ci-C₂o alkyl nitrite is isopentyl nitrite.

7. The substrate according to claim 4, wherein the organic nitrite is a C₃-Ci₂ alkenyl nitrite.

8. The substrate according to claim 7, wherein the C₃-C₁₂ alkenyl nitrite is isoprenyl nitrite.

9. The substrate according to claim 4, wherein the organic nitrite is a C₃-C_{] 2} alkynyl nitrite.

10. The substrate according to claim 4, wherein the organic nitrite is an aryl nitrite.

1 1. The substrate according to any one of the preceding claims, wherein the substrate is formed from one or more materials selected from the group consisting of metal, synthetic polymer, biopolymer, glass, and ceramic.

12. The substrate according to any one of the preceding claims, wherein the substrate is a medical device, an implant, a water treatment device or part thereof, a food packaging container, a food handling device or part thereof, an industrial pipe or a water tower.

13. The substrate according to claim 12, wherein the substrate is a medical device.

14. The substrate according to claim 13, wherein the medical device is a replacement joint, a urinary catheter, a percutaneous access catheter, and endotracheal tube, a stent, a pacemaker, a prosthetic, a bandage, a wound dressing or a contact lens.

15. A substrate comprising a bacteriostatic surface, the bacteriostatic surface comprising a nitrite containing polymer.

16. The substrate according to claim 15, wherein the nitrite containing polymer is a nitrite containing plasma polymer.

17. The substrate according to claim 16, wherein the nitrite containing plasma polymer is prepared by plasma polymerisation of one or more organic nitrites.

18. The substrate according to claim 17, wherein the organic nitrite is a Cj -C₂o organic nitrite.

19. The substrate according to claim 18, wherein the organic nitrite is a C]-C₂₀ alkyl nitrite.

20. The substrate according to claim 19, wherein the Ci-C₂o alkyl nitrite is isopentyl nitrite.

21. The substrate according to claim 18, wherein the organic nitrite is a C₃-Ci₂ alkenyl nitrite.

22. The substrate according to claim 21, wherein the C3-C12 alkenyl nitrite is isoprenyl nitrite.

23. The substrate according to claim 18, wherein the organic nitrite is a C₃-C₁₂ alkynyl nitrite.

24. The substrate according to claim 18, wherein the organic nitrite is an aiyl nitrite.

25. The substrate according to any one of claims 15 to 25, wherein the substrate is formed from one or more materials selected from the group consisting of metal, synthetic polymer, biopolymer, glass, and ceramic.

26. The substrate according to any one of claims 15 to 25, wherein the substrate is a medical device, an implant, a water treatment device or part thereof, a food packaging container, a food handling device or part thereof, an industrial pipe or a water tower.

27. The substrate according to claim 26, wherein the substrate is a medical device.

28. The substrate according to claim 27, wherein the medical device is a replacement joint, a urinary catheter, a percutaneous access catheter, an endotracheal tube, a stent, a pacemaker, a prosthetic, a bandage, a wound dressing or a contact lens.

29. A process for preparing a substrate comprising an antibacterial and/or bacteriostatic surface, the process comprising exposing a substrate and an organic nitrite to a plasma environment under conditions to deposit a nitrite containing plasma polymer on the surface.

30. The process according to claim 29, wherein the nitrite containing plasma polymer is prepared by plasma polymerisation of one or more organic nitrites.

31. The process according to claim 30, wherein the organic nitrite is a Ci-C₂o organic nitrite.

32. The process according to claim 3 1 , wherein the organic nitrite is a C₁-C₂₀ alkyl nitrite.

33. The process according to claim 32, wherein the Ci-C₂o alkyl nitrite is isopentyl nitrite.

34. The process according to claim 31 , wherein the organic nitrite is a C₃-Ci₂ alkenyl nitrite.

35. The process according to claim 34, wherein the C₃-C₁₂ alkenyl nitrite is isoprenyl nitrite.

36. The process according to claim 31 , wherein the organic nitrite is a C₃-Ci₂ alkynyl nitrite.

37. The process according to claim 3 1 , wherein the organic nitrite is an aryl nitrite.

38. The process according to any one of claims 29 to 37, wherein the substrate is formed from one or more materials selected from the group consisting of metal, synthetic polymer, biopolymer, glass, and ceramic.

39. The process according to any one of claims 29 to 38, wherein the plasma deposition step comprises exposing the substrate and the organic nitrite to low power glow plasma discharge under continuous power and then exposing the substrate and the organic nitrite to pulsed low power glow plasma discharge.

40. The process according to claim 39, wherein the organic nitrite is plasma deposited in a continuous deposition step for a period of from about 1 to about 15 minutes and then in a pulsed deposition step comprising a pulsed deposition rate of from about 1/10 ms to about 1/30 ms for a total period of from about 10 to about 120 minutes.

41. A method for preventing or inhibiting bacterial growth on a surface of a substrate, the method comprising forming an antibacterial and/or bacteriostatic surface comprising a nitrite containing polymer on the substrate and exposing the substrate to an environment containing water that is susceptible to bacterial infection.

42. The method according to claim 41, wherein the nitrite containing polymer is a nitrite containing plasma polymer.

43. The method according to claim 42, wherein the nitrite containing plasma polymer is prepared by plasma polymerisation of one or more organic nitrites.

44. The method according to claim 43, wherein the organic nitrite is a Ci-C₂o organic nitrite.

45. The method according to claim 44, wherein the organic nitrite is a C1-C20 alkyl nitrite.

46. The method according to claim 45, wherein the Ci-C₂o alkyl nitrite is isopentyl nitrite.

47. The method according to claim 44, wherein the organic nitrite is a C3-C12 alkenyl nitrite.

48. The method according to claim 47, wherein the C3-C 12 alkenyl nitrite is isoprenyl nitrite.

49. The method according to claim 44, wherein the organic nitrite is a C3-C12 alkynyl nitrite.

50. The method according to claim 44, wherein the organic nitrite is an aryl nitrite.

51. The method according to any one of claims 41 to 50, wherein the substrate is formed from one or more materials selected from the group consisting of metal, synthetic polymer, biopolymer, glass, and ceramic.

52. The method according to any one of claims 41 to 51 , wherein the substrate is a medical device, an implant, a water treatment device or part thereof, a food packaging container, a food handling device or part thereof, an industrial pipe or a water tower.

53. The method according to claim 52, wherein the substrate is a medical device.

54. The method according to claim 53, wherein the medical device is a replacement joint, a urinary catheter, a percutaneous access catheter, an endotracheal tube, a stent, a pacemaker, a prosthetic, a bandage, a wound dressing or a contact lens.

55. A method for selectively inhibiting or preventing the growth of bacterial cells on a substrate when it is used in the presence of eukaryotic cells, the method comprising forming an antibacterial surface comprising a nitrite containing polymer on the substrate and exposing the substrate to an environment containing water that is susceptible to bacterial infection.

56. The method according to claim 55, wherein the nitrite containing polymer is a nitrite containing plasma polymer.

57. The method according to claim 56, wherein the nitrite containing plasma polymer is prepared by plasma polymerisation of one or more organic nitrites.

58. The method according to claim 57, wherein the organic nitrite is a Ci-C₂o organic nitrite.

59. The method according to claim 58, wherein the organic nitrite is a C₁-C₂₀ alkyl nitrite.

60. The method according to claim 59, wherein the Ci-C₂o alkyl nitrite is isopentyl nitrite.

61. The method according to claim 58, wherein the organic nitrite is a C3-C₁₂ alkenyl nitrite.

62. The method according to claim 61 , wherein the C3-C₁₂ alkenyl nitrite is isoprenyl nitrite.

63. The method according to claim 58, wherein the organic nitrite is a C3-C₁₂ alkynyl nitrite.

64. The method according to claim 58, wherein the organic nitrite is an aryl nitrite.

65. The method according to any one of claims 55 to 64, wherein the substrate is formed from one or more material selected from the group consisting of metal, synthetic polymer, biopolymer, glass, and ceramic.

66. The method according to any one of claims 55 to 65, wherein the substrate is a medical device, an implant, a water treatment device or part thereof, a food packaging container, a food handling device or part thereof, an industrial pipe or a water tower.

67. The method according to claim 66, wherein the substrate is a medical device.

68. The method according to claim 67, wherein the medical device is a replacement joint, a urinary catheter, a percutaneous access catheter, an endotracheal tube, a stent, a pacemaker, a prosthetic, a bandage, a wound dressing or a contact lens.