WO2012035302A1

WO2012035302A1 - Binding and non-binding polymers

Info

Publication number: WO2012035302A1
Application number: PCT/GB2011/001354
Authority: WO
Inventors: Mark Bradley; Maurice Patrick Gallagher; Salvatore Pernagallo; Mei Wu
Original assignee: The University Court Of The University Of Edinburgh
Priority date: 2010-09-16
Filing date: 2011-09-16
Publication date: 2012-03-22
Also published as: GB201015492D0

Abstract

Polyacrylate and polyurethane polymers that are selected to have either; surface properties that allow microorganisms, in particular bacteria, to bind to the surface; or have surface properties that are repellent to the binding of microorganisms are described. Products comprising, consisting of or coated with the polymers are also described. The polyacrylate polymers include polyacrylates that are non-binding or selectively binding to microorganisms and have a methacrylate or acrylate containing polymer backbone that is provided with pendant tertiary amine groups.

Description

Binding and Non-Binding Polymers

Field of the Invention

The present invention relates to the provision of polymers that are selected to have either; surface properties that allow microorganisms, in particular bacteria, to bind to the surface; or have surface properties that are repellent to the binding of microorganisms. Methods for identifying suitable polymers are provided. Products comprising, consisting of or coated with the polymers are also provided. Background to the Invention

It is well known that the microbial surface or secreted components from microbes are determinant factors in the surface adhesion process. Other physicochemical features such as pH, temperature, composition of surrounding medium and surface conditioning factors are also known to affect surface attachment.¹

In order to control bacterial or other microorganism attachment, there is a need for materials to which a microorganism of interest demonstrates attachment or repulsion (binding or non-binding properties). These materials when discovered could underpin wide-ranging applications in hygiene and avoidance of bio-fouling, in encapsulation or detection systems, or in nano/micro-fabrication or synthetic biology.

Materials which bind microorganisms, can be used for the selective capture of bacteria, spores or viruses on cleaning materials used in clinical, industrial and domestic environments and could offer, for example a means for the rapid isolation of hospital pathogens if selective binding can be achieved. They could also provide opportunities for innovative intervention approaches, such as the selective reduction of pathogen loads in animals by application via animal feeds. As a further example purification of a fluid could be achieved by contacting the fluid with an article that includes a microorganism binding surface. Conveniently the fluid could be passed through a filter that included a binding surface or surfaces.

Materials which repel microorganisms could be used to minimise surface contamination through the development of microbe repelling surfaces for use in a wide range of applications. For example work surfaces in clinical and food handling environments, in the domestic environment (e.g. in shower fitments), the surfaces of medical devices (e.g. surgical implants, orthodontic devices) and more generally any article that may be used in any environment that includes undesirable (or harmful) microorganisms. Packaging for foodstuffs that resists adherence by bacteria is a further example. A yet further example would be the use of components resistant to microorganism attachment in nano- or micro fabricated platforms or molecular or cellular 'machines'. This would avoid contamination by microorganisms resulting in loss of function of the nano- or micro fabricated article or provide a method for location specific bioassembly.

Description of the Invention

Polymer microarrays have become established as a method to identify polymers that can enrich, manipulate or modulate a variety of adherent or suspended mammalian cell types, including stem cells for regenerative medicine or tissue engineering applications.^2"10 The present invention is based on the identification (by use of polymer microarrays) of polymers, especially polyacrylate and polyurethane, that exhibit distinctive binding or non-binding properties towards microorganisms, in particular bacteria. The polymers can be selective in their binding properties, for example binding a particular type of bacterium in preference to or even whilst being repellent to another.

Thus the present invention provides a method for identifying polymers binding to or repelling a microorganism comprising:

preparation of a library of polymer samples;

exposing the polymer samples to a target microorganism; and

observing binding or non-binding of the target microorganism to the polymer samples.

Advantageously the polymer samples are made by high throughput methods such as parallel synthesis techniques or inkjet printing. Advantageously the testing is carried out by preparing micro-arrays of polymer samples which are then exposed to the microorganism.

The polymers can have a number of uses. The polymers provided in the following description may be used in a number of applications where the ability to bind to microorganisms or to repel or at least only weakly bind to microorganisms is useful. The organisms may be harmful or non- harmful bacteria, fungi, protozoans, spores or viruses, for example. The microorganisms may be alive or dead. For example the polymers may bind or repel dead bacteria cells as well as live cells.

The present invention provides uses (i.e. methods of using) the polymers described herein including methods of binding microorganisms to a substrate e.g. the surface of a substrate and methods of preventing binding of microorganisms to a substrate e.g. the surface of a substrate. Selective binding of particular organisms may also be achieved. The methods can be applicable in a wide range of technologies including microbial detection or filter systems, water purification, delivery and treatment; medical devices and appliances; food industry and related technologies.

Thus the present invention provides an article comprising, consisting of, consisting essentially of or coated with a microorganism binding polymer or a microorganism non- binding polymer as described herein. Thus the present invention provides a coating for a substrate, the coating comprising, consisting of or consisting essentially of a microorganism binding polymer or a microorganism non-binding polymer as described herein.

The present invention also provides microorganism binding polymers or microorganism non-binding polymers as described herein. The polymers may be used in manufacture of an article or a coating for a substrate. Such polymers may also include antimicrobial agents or enzymatic substrates within the polymers.

For example the article may be a medical device such as an endotracheal tube or a stent or a replacement joint or replacement joint component used for temporary or permanent placement in a patient. The article includes a non-binding polymer, for example as a coating or for further example as the material from which a tubing wall is constructed. By virtue of the non-binding properties of the polymer the article exhibits resistance to becoming colonised by harmful bacteria or other microorganisms. Thus biofilm formation and infection is reduced or prevented. As well as uses for medical devices such as tubes (e.g. catheters), stents, replacement joints and other implants, including dental implants, other medical uses for non-binding polymers can include use in wound dressings and surgical gauze, where the polymer can be used to prevent or resist infection (colonisation by microorganisms) of the dressing and hence the wound covered by the dressing. Other uses can include in contact lenses, to aid in reducing eye infections, tooth coatings or implants, surgical (or other) face masks and protective body suits, surgical and imaging devices, such as endoscopes, and surgical tools (e.g. dissection kits). For dental healthcare non-binding polymers may be used for materials for dental hygiene or treatment e.g. bacteria repellent tooth and gum care in toothpastes, mouthwashes, denture coatings, in the prevention of acid erosion, and for delivery of remineralisation agents. Other uses may be in coatings for tooth or bone implants. The non-binding or selectively binding polymers described herein may find particular oral hygiene use against plaque colonising bacteria such as Streptococci - especially S.gordonii, S.mitis, S. oralis; and Actinomyces esp. A.naeslundii; Neisseria esp. N. mucosa.

For further example the article may be a work surface or a tool used in a hospital, clinic, or domestic situation. For example, in a catheter, a food preparation area such as a kitchen, or a shower or in or on clothing. . Again use of one of the non-binding polymers described herein can avoid the build up of a microbial agent to unwanted levels. Equally, the article could be a platform for molecular or cellular fabrication, where a microorganism is prevented from binding in particular locations on the platform.

In general the non-binding polymers may be used to avoid colonisation or fouling by microorganisms. For further example the polymers may be used in methods of preventing or avoiding fouling of water systems such as water supply systems, heating and cooling units, and storage tanks. Colonisation by microorganisms will be resisted by making use of the non-binding polymers as coatings or constituents of pipes, pumps storage tanks and the like, plaster, paints, and other construction fabrication materials or other surface coatings. Paints and other coatings applications may include protective / anti-biofouling coatings for marine or other architecture (e.g. ships, wind turbines, wave power devices) as well as coatings for use in water tanks and systems. Yet further uses of the non-binding (repellent) or selectively binding polymers can be envisaged for consumer products (from personal hygiene and household cleaning to food stuffs). These can include polymer containing products for topical use (e.g. bacteria repellent hand cream), use of non-binding (repellent) polymers in household cleaning products and repellent food packaging.

Binding polymers may be used for example the article may be a filter for water or biological fluids. The filter includes filter media comprising, consisting, consisting essentially of, or coated with a binding polymer of the invention. On passage of a fluid contaminated with an undesired microorganism e.g. Salmonella such as S. enteritidis or S. typhimurium; Campylobacter such as Campylobacter jejuni; or E. coli, the contaminating microorganism becomes attached to the binding polymer. Thus the fluid is purified.

For example the article may be a swab including a binding polymer and used to swab areas that may be contaminated. The swab can be tested, directly or indirectly for the presence of microbes or their components. The swab can assist in disinfecting the area by virtue of the binding of contaminating microbes. Thus the swab can disinfect an area, for example a work surface or a wound in a human or animal patient. Wound dressings may also be made that include a binding polymer. These can assist in disinfecting a wound and they can be tested for the presence of microbes of interest in a similar fashion. For further example the article may be a cleaning material such as a cloth or a liquid (that may be a suspension or a solution) that contains binding polymers for rapid scavenging of microorganisms such as bacteria. Selective binding polymers can be used to collect or scavenge harmful microorganisms (e.g. bacteria) whilst leaving non harmful or beneficial microorganisms unaffected or relatively unaffected .

For further example, the article may be a contained or slow release structure for use in agriculture, or in human or animal subjects. Such articles may be used for bio-control, probiotic delivery, use in vaccination or therapy, or for bioconversion or bio-production. Micro-organisms adhered to a binding polymer surface in such an article may themselves, or components from them, be released gradually - thus providing controlled release therapy in a human or animal subject, for example.

Where the polymers show binding to bacteria, the bacteria typically appear firmly attached to the polymer surface forming single cell attachments or closely packed arrangements (small or micro- colonies), possibly indicating early or mature biofilm formation. In contrast the non-binding polymers showed little attachment of the subject bacteria and no tendency for biofilm formation was observed. In one embodiment of the invention polyacrylate polymers that show strong binding to bacteria are formed in a polymerisation reaction where one of the monomers is a hydroxyalkyl methacrylate monomer. The alkyl group of the hydroxyalkyl function may have from 1 to 10 or even from 1 to 4 carbon atoms. The hydroxyalkyl methacrylate monomer may be for example selected from HBMA (Hydroxybutylmethacrylate - typically available as a mixture of isomers but a single isomer could be used if desired) or HEMA (2-hydroxyethylmethacrylate). The hydroxyalkyl methacrylate may be copolymerised with other acrylate monomers or other unsaturated monomers. The range of molar ratio of hydroxyalkyl methacrylate monomer to other monomers used may be from - 95:5 to 45:55 - or even 90:10 to 50:50. Higher quantities of other monomers may be used in some circumstances.

Examples of suitable monomers for copolymerisation with a hydroxyalkyl methacrylate include DEAA [diethylacrylamide], D AEMA [2-(dimethylamino)ethyl methacrylate], BACOEA (2-[[(butylamino)carbonyl]oxy]ethyl acrylate) and VI [1-vinylimidazole].

Alternative compositions found to bind bacteria include acrylate polymers made by polymerisation of MEMA (2-methoxyethylmethacrylate) or BMA (butyl methacrylate) with other monomers to form combinations such as:

MEMA with A-H (acrylic acid) and DEAEA [2-(diethylamino)ethyl acrylate];

MEMA with DMVBA (Ν,Ν-Dimethylvinylbenzylamine);

MEMA with EGMP-H (Ethylene glycol methacrylate phosphate);

BMA with DEAEA [2-(diethylamino)ethyl acrylate]; and

BMA with DMAEA [2-(dimethylamino)ethyl acrylate]. Yet further compositions including MEMA that bind to bacteria include two where the MEMA is polymerised with GMA (glycidyl methacrylate), with the epoxide function of the GMA being subsequently reacted with an amine: - for example DnBA (Di-n- butylamine) or TMPDA (trimethylpropane diamine). An excess of a secondary amine is used to react all the glycidyl (epoxy) functions of the GMA as illustrated and discussed further hereafter and with reference to scheme 2.

Certain acrylate polymers have been shown for example to bind well to two different types of bacteria, Salmonella and E. coli, in particular to the major food borne pathogen Salmonella enterica serovar typhimurium (S. typhimurium) and a laboratory strain of E. coli, with the binding affinity varying with polymer structure.

Preferred compositions making use of the above monomer mixtures are listed in Table 1 below. Results of binding assays with S. typhimurium and E. coli are discussed hereafter. As is known by the skilled person terms such as good binding and poor binding are relative and will depend on the bacterial growth conditions and media, the bacterial strain and the manner and place in which the polymer is applied. The testing regime applied is described hereafter in the Detailed Description of the Invention. Table 1 - Polyacrylates binding to S. typhimurium and E. coli

PA181 has also been shown to bind to S. enteritidis. However, when PA296 was tested against S. enteritidis, it showed low binding affinity (repellence) demonstrating that polymer compositions can show selectivity, even between different bacteria strains such as between Salmonella strains.

Acrylate compositions binding to S. typhimurium and S. enteritidis but with low binding to E. coli are listed in Table 1 a below. These two polymers show selective affinity to Salmonella strains rather than to E. coli.

Table 1a

Polyacrylates binding to S. typhimurium and S. enteritidis but with low binding to

E. coli

Monomers of Table 1 a:

MEMA: 2-methoxyethyl methacrylate;

HBMA: hydroxybutylmethacrylate;

VP-2: 2-vinylpyridine;

DMAA: dimethylacrylamide.

Acrylate compositions binding E. coli but with low binding to S. typhimurium are listed in Table 1 b below. Both these compositions include GMA with the epoxy function reacted with an amine as discussed above for GMA containing compositions of Table 1.

Table 1 b

Polyacrylates binding to E. coli but with low binding to S. typhimurium

Monomers of Table 1 b: MMA: methyl methacrylate;

GMA: glycidyl methacrylate;

DBnA: dibenzylamine;

Mpi: 1-methylpiperazine.

In another embodiment the invention provides polyurethane polymers that are binding to bacteria and are polyurethane polymers formed by polymerising a polydiol with a di- isocyanate and optionally with an extender molecule, such as a diol. The extender molecules have the effect of modifying the physical character of the polymers, for example, polymer shape, viscosity and polymer state. Such polyurethane polymers have been found to bind to both E. coli and Salmonella, and in particular S. typhimurium.

The polydiol may be selected from the group consisting of:

PEG: poly(ethylene glycol);

PPG: poly(propylene glycol);

PTMG: poly(tetramethylene glycol) also known as poly(butylene glycol);

PHNGAD: poly[1 ,6-hexanediol/neopentyl glycol/diethylene glycol-alt-(adipic acid)]diol; and

PHNAD: poly[1 ,6-hexanediol/neopentyl glycol-alt-(adipic acid)]diol.

PPG: poly(propylene glycol) and PTMG: poly(butylene glycol) have shown particularly good results in certain polymer compositions. The molecular weights of the polydiol may be from Mn=200 to Mn=1500 or higher.

The di-isocyanate may be selected from the group consisting of:

HDI: 1 ,6-diisocyanohexane;

MDI: 4,4'-methylenebis(phenylisocyanate);

TDI: 4-methyl-1 ,3-phenylene diisocyanate;

PDI: 1 ,4-diisocyanobenzene;

HMDI: 4,4'-methylenebis(cyclohexylisocyanate); and

BICH: 1 ,3-bis(isocynanatomethyl)cyclohexane.

Suitable extenders include: BD: 1 ,4-butanediol;

EG: ethylene glycol;

PG: propylene glycol;

ED: ethylene diamine;

OFHD: 2, 2, 3,3,4,4,5, 5-octafluoro-1 ,6-hexanediol;

DEAPD: 3-diethylamino-1 ,2-propanediol;

DMAPD: 3-dimethylamino-1 ,2-propanediol; and

NMPD: 2-nitro-2-methyl-1 ,3-propanediol. Preferred compositions of polyurethane polymers exhibiting binding to bacteria are listed in Table 2 below. Results of binding assays are discussed hereafter.

Table 2

Polyurethanes binding to bacteria

PU Polymer Structure

reference Ratio (mol)

Diol Mn Dis. Ext.

number Diol Dis Ext

PU39 PT G 2000 HDI BD 0.25 0.52 0.23

PU92 PTMG 1000 HDI BD 0.485 0.515 0

PU104^A PHNGAD 1800 MDI DEAPD 0.25 0.52 0.23

PU116 PPG 425 BICH BD 0.485 0.515 0

PU118^A PPG 425 MDI DMAPD 0.25 0.52 0.23

PU119^A PPG 1000 MDI DMAPD 0.25 0.52 0.23

PU120 PPG 425 BICH DEAPD 0.25 0.52 0.23

PU126^A PPG 425 TDI DMAPD 0.25 0.52 0.23

PU138^A PTMG 250 BICH EG 0.25 0.52 0.23

PU159 PTMG 250 MDI BD 0.25 0.52 0.23

PU171 ^A PTMG 250 HDI none 48.5 51.5 0

PU178 PTMG 1000 HDI NMPD 0.25 0.52 0.23

PU185^A PHNAD 900 MDI OFHD 0.17 0.52 0.33

PU186^A PTMG 650 BICH OFHD 0.25 0.52 0.23

PU208^A PPG 1000 MDI OFHD 0.25 0.52 0.23

PU219^A PHNAD 900 BICH DMAPD 0.25 0.52 0.23

PU220^A PHNAD 900 MDI DMAPD 0.25 0.52 0.23

PU222^A PHNAD 900 BICH OFHD 0.25 0.52 0.23 [Diol = polydiol as listed above; Mn= molecular weight (number average) of polydiol; Dis=diisocyanate; Ext. = extender]

The polymers marked ^Λ show good binding to S. enteritidis, S. typhimurium and E. coli. The other polymers showed good binding to S. typhimurium and E. coli.

The structure of the polyurethanes is illustrated schematically in Scheme 1 below which shows PL) 178 and PU222 from Table 2 as examples.

Scheme 1

R=-CH2(CH₃)₄CH_2- Molecular ratio:

*CH2C(CH3)2CH2~ PU178: PT G/HDI/NMPD (25/52/23)

PU222: PHNAD/BICH/OFHD (25/52/23)

Polyurethanes with low binding to S. enteritidis are also provided in Table 2a below. Selectivity in binding to different organisms can also be provided.

Table 2a

Polyurethanes with poor binding to S. enteritidis

[Diol = polydiol as listed above; Mn= molecular weight (number average) of polydiol; Dis=diisocyanate; Ext. = extender]. The polymer marked ^Λ (PU163) shows poor binding to S. enteritidis but strong binding to S. typhimurium and E. coli.

The other polymers showed poor binding to S. enteritidis but were not tested against S. typhimurium and E. coli.

Other polyurethanes with selective binding are listed in Table 2b below.

Table 2b

Polyurethanes with selective binding

Binding Character:

a= binding to S. enteritidis but not tested against S. typhimurium and E. coli;

b= binding to S. enteritidis and non binding to S. typhimurium and E. coli;

c= binding to S. enteritidis and S. typhimurium and non binding to E. coli;

d= binding to E. coli, non binding to S. enteritidis and S. typhimurium;

e= non binding to all three bacteria tested.

From the above table it can be seen that polyurethanes such as PU1 , PU4, PU161 and PU65 including ethylene glycol (as polydiol - PEG - or as extender - EG) can show good non-binding (repellence) properties or selective binding.

In another embodiment acrylate polymers that are repellent to bacteria, for example E. coli or Salmonella, in particular to S. typhimurium and S. enteritidis are provided.

The polymers may be formed by the copolymerisation of MMA (methyl methacrylate) and GMA (glycidyl methacrylate), with the epoxide function of the GMA being reacted with a secondary amine. An excess of a secondary amine is used to react all the glycidyl (epoxy) functions of the GMA to produce polymers with a hydroxy function beta to the nitrogen of the amine as shown schematically in Scheme 2 below. The ratio of M A to GMA employed may be from 95:5 to 45:55 or even 90:10 to 50:50. Even higher GMA content may be employed. Suitable examples of secondary amines are listed in Table 3 below and include dialkyl amines, alky/aryl amines and cyclic amines such as pyrrole.

As an alternative and as shown in Scheme 2 (with reference to the examples of polymers PA235 and PA236 of Table 3) a tertiary amine function is present in an acrylate monomer used to form the polymer and may be attached via an ester linkage to the polymer backbone, with the ester oxygen beta to the nitrogen. As can be seen in Table 3a where this approach is employed the polymers may comprise other unsaturated or acrylate or methacrylate monomers in addition to MMA. For example styrene (St), methacrylic acid (MA-H) or butyl methacrylate (BMA).

Scheme 2 le

Preferred compositions showing poor binding to bacteria are listed in Table 3 with the amine used to react with the glycidyl function of GMA, where present, listed. Results of binding experiments are discussed in more detail hereafter. Compositions marked ^Λ showed low binding (repellence for three strains - S. enteritidis, S. typhimurium and E. coli).

The polymers marked ^ΛΛ show very poor binding to S. enteritidis but were not tested against S. typhimurium and E. coli.

The other polymers showed poor binding to S. typhimurium and variable binding to E. coli but were not tested against S. enteritidis, except for PA306, PA323, PA426 and PA327. Polymers PA326, PA323, and PA426 showed relatively weak binding to S. enteritidis and PA327 was non-binding to the same bacterium.

Table 3

Polyacrylates exhibiting poor binding to bacteria (repellence)

Monomers of able 3:

MMA: methyl methacrylate; BMA: butyl methacrylate;

MEMA: 2-methoxyethylmethacrylate;

HEMA: 2-hydroxyethylmethacrylate;

DMAE A: 2-(dimethylamino)ethyl methacrylate;

DEAEMA: 2-(diethylamino)ethyl methacrylate;

DEAEA: 2-(diethylamino)ethyl acrylate;

DMAPMAA: dimethylaminopropyl methacrylamide;

DMVBA: N,N-Dimethylvinylbenzylamine;

DMAEA: 2-(dimethylamino)ethyl acrylate;

EGMP-H: Ethylene glycol methacrylate phosphate;

St: styrene;

GMA: Glycidyl methacrylate;

A-H: acrylic acid;

MA-H: Methacrylic acid.

Amines of Table 3:

DnBA: Di-n-Butylamine;

cHMA: cyclohexanemethylamine;

Bn A: N-benzylmethylamine;

AEPy: 2-(2-Methylaminoethyl) pyridine;

Pyrle: Pyrrole;

MAn: N-methylaniline.

In some examples of polyacrylate polymers of the types such as shown in Table 3 selectivity between types of bacteria is observed.

Compositions not binding to either of the two Salmonella strains tested but binding to E.coli are listed in Table 3a below. In the polymers including GMA the third (amine) component of the composition is added in sufficient quantity to react with all the epoxy groups, as discussed above. Table 3a

Acrylates with low binding to S. typhimurium and S. enteritidis but binding to E.

coli.

Monomers of Table 3a:

MMA: methyl methacrylate;

MEMA: 2-methoxymethyl methacrylate;

GMA: glycidyl methacrylate;

DEAEMA: 2-(diethylamino)ethyl methacrylate;

DBnA: dibenzylamine;

Pyrle: pyrrole;

MAn: N-methylaniline;

MA: methyl acrylate;

DAAA: diactetone acrylamide.

Polyacrylates that showed strong binding to S. enteritidis are listed in Table 3b below Compositions showing low binding to E. coli and S typhimurium but binding to S. enteritidis are also provided. Table 3b

Polyacrylates exhibiting strong binding to S. enteritidis

Monomers of Table 3b:

MMA: methyl methacrylate;

MEMA: 2-methoxymethyl methacrylate;

GMA: glycidyl methacrylate;

HBMA: hydroxybutylmethacrylate;

MEA: methoxyethyl acrylate;

HBMA: hydroxybutylmethacrylate;

PAA: N-isopropylacrylamide;

MA-H: methacrylic acid;

EGMP-H: ethylene glycol methacrylate phosphate;

A-H: acrylic acid;

DEAEA: 2-(diethylamino)ethyl acrylate;

BnMA: N- benzylmethylamine.

The polymer marked ^Λ showed strong binding to S. enteritidis but poor binding to S. typhimurium and E. coli.

The other polymers showed strong binding to S. enteritidis but were not tested against S. typhimurium and E. coli. Compositions showing low binding to E.coli and S. enteritidis but binding to typhimurium are shown in table 3c below.

Table 3c

Acrylates with low binding to E. coli and S. enteritidis but binding to S.

typhimurium

Monomers of Table 3c:

MEMA: 2-methoxymethyl methacrylate;

HEMA: 2-hydroxyethyl methacrylate;

MTEMA: 2-(methylthio)ethyl methacrylate;

DEAEA: 2-(diethylamino)ethyl acrylate;

A-H: acrylic acid.

Thus the present invention provides polyacrylate polymers that are selectively binding or non-binding (repellent) to microorganisms, in particular bacteria, said acrylate polymers comprising tertiary amine functions of the form:

wherein P represents an acrylate or methacrylate containing polymer backbone and R' and R" are each independently selected from:

alkyl groups (for example C1 to C10) that may be saturated or unsaturated and may be substituted;

aryl or heteroaryl groups ( for example C5 - C15) that may be substituted; or

R' and R" are fused to form a ring (for example C4 to C10) that may be saturated or unsaturated, may contain heteroatoms and may be substituted. Thus for example polymers comprising MEMA or MMA and a second monomer that either contains the tertiary amine function or than can be reacted with a secondary amine to give the tertiary amine function (e.g. GMA) give good results as listed in the tables above and shown in the results discussed hereafter. The polymers may include a further, optional monomer or monomers. The polymer backbone P has a number of the pendant tertiary amine functional groups depending on the amount of second monomer employed.

Thus the present invention provides acrylate polymers that are non-binding (repellent) or selectively binding to microorganisms, in particular bacteria, said acrylate polymers comprising MEMA or MMA and a second monomer that either contains a tertiary amine function or that can be reacted with a secondary amine to give a tertiary amine function (e.g. GMA or another epoxy functionality). Where MMA is employed with GMA and without other monomer for building the polyacrylate backbone the molar ratio of MMA to GMA may be from 95:5 to 40:60 or even 90:10 to 50:50. The GMA is then reacted with sufficient amine to react with all the glycidyl groups present. Where MEMA is employed with GMA similar ratios may be employed

More generally the present invention provides polyacrylate polymers that are selectively binding or non-binding (repellent) to microorganisms, in particular bacteria, said acrylate polymers having a methacrylate or acrylate containing polymer backbone with pendant tertiary amine groups of the structure;

X

I

R ^R¹

wherein X is a linker group bonding to the polymer backbone selected from the group consisting of substituted or unsubstituted alkylene that may be cyclic and may be unsaturated (for example C1 -C10 or even C1-C4); substituted or unsubstituted aryl or heteroaryl; and

R' and R" are each independently selected from:

aryl or heteroaryl groups ( for example C5 - C15) that may be substituted; or R' and R" are fused to form a ring (for example C4 to C10) that may be saturated or unsaturated, may contain heteroatoms and may be substituted.

The linker groups X are typically bonded to the polymer backbone via an ester or amide connection, generally ester, of a methacrylate or acrylate monomer used in the preparation of the polymer. However other bonding of the groups X to the polymer backbone may be envisaged, for example directly to a carbon chain in the polymer backbone. Typically about 5% to 60%; or even about 10% to 60%; of the monomers forming the polymer backbone will be functionalised with the pendant tertiary amine groups. However polymers having even higher pendant tertiary amine group content may be envisaged for some applications. By alkyl is meant herein a hydrocarbyl radical, which may be straight-chain, cyclic or branched (typically straight-chain unless the context dictates to the contrary). An alkylene group is a diradical formed formally by abstraction of a hydrogen atom from an alkyl group. Where groups X, R' and R" are substituted, they may be substituted, for example once, twice, or three times, e.g. once, i.e. formally replacing one or more hydrogen atoms of the alkylene, alkyl, aryl or heteroaryl group. Examples of such substituents are hydroxy, halo (e.g. fluoro, chloro, bromo and iodo),SF₅, CF₃ ,aryl, aryl hydroxy, nitro, amino, alkoxy, alkylthio, carboxy, cyano, thio, formyl, ester, acyl, thioacyl, amido, sulfonamido, carbamate and the like. Where the substituent is amino it may be NH₂, NHR or NR₂, where the substituents R on the nitrogen may be alkyl, aryl or heteroaryl (for example substituted or unsubstituted C1 -C20 or even C1 -C10).

It will readily be appreciated by the skilled person that the selectively binding polymers will bind only to certain microorganisms and are therefore non-binding with respect to other microorganisms. Therefore for use in the methods described herein selectively binding polymers may be usefully employed in applications where non-binding behaviour is required. The polymer can be used in "non-binding" methods and applications where the microorganisms that do bind to the polymer are not likely to be present or are not considered particularly harmful or detrimental. Furthermore if a particular microorganism exhibits relatively weak binding to a polymer (e.g a binding level of up to say 20,000 bacteria/mm², or thereabouts) this may not prevent the use of the polymer in a particular non-binding application even where the microorganism bound.

Brief description of the Drawings

Figure 1 shows the results of fluorescence imaging of polymer arrays; and

Figures 2 and 3 show tables of results for testing polymers against a range of bacteria. Detailed Description of the Invention with reference to the preparation of some exemplary compositions of the invention and results of tests

MATERIALS AND METHODS FOR PREPARATION OF POLYMERS AND FABRICATION OF MICROARRAYS

All chemicals were of analytical grade and used as received without further purification. Silane-prep glass slides, tetracycline, sodium cacodylate trihydrate and all the monomers used were from Sigma-Aldrich. Phosphate buffered saline (PBS) tablets were from Oxoid. GeneFrames were bought from Thermo Scientific, and 2.5% (w/v) glutaraldehyde and 1 % (w/v) osmium tetroxide were purchased from Electron Microscopy Sciences. The rectangular four-well plates were obtained form Nunc. Gridded glass coverslips were purchased from CELL-VU.

METHODS

Polymer microarray fabrication:⁷' ¹¹

Polymer microarrays were fabricated by contact printing (QArraymini, Genetix, UK) with 32 aQu solid pins (K2785, Genetix) using 1 % w/v polymer solutions in N- methylpyrrolidone (NMP) placed in polypropylene 384-well microplates. The printing conditions used were 5 stamps per spot, with a 200 ms inking time and a 100 ms stamping time on saline treated slides and coated with agarose Type l-B (Sigma- Aldrich, UK).

Polymer manufacture:

(1 ) Polyacrylates: The polyacrylates were made by parallel synthesis on a millimolar scale via a typical free radical polymerisation.

The monomers (two or three as required) were mixed together in selected proportions together with suitable solvents, for example toluene. Reaction was initiated with a free radical generator (AIBN) and heat. The solid polymers were precipitated by dropwise addition into a non-polar solvent (hexane, cyclohexane, diethyl ether, or a mixture thereof) and collected by centrifugation. Typical Mw results for the polyacrylates were of the order of 70,000 to 320,000 Daltons, except for those prepared using G A as a monomer for subsequent functionalisation with a secondary amine to make tertiary amine pendant group. These generally had a higher Mw e.g. above 2,000,000 Daltons. However polymers of either type may be prepared with different Mw profiles by appropriate adjustment of the production method as known to those skilled in the art.

Where GMA was used as a monomer, the functionalisation of the epoxide group with an amine was carried out following preparation of the base polymer. The reaction is carried out in a suitable solvent, for example cyclohexanol/xylene mixtures. Reaction was carried out with heat (e.g. 130°C) and in the presence of silica gel (e.g. silica gel 60) as catalyst. Essentially complete conversion of the epoxide functions to tertiary amine could be achieved.

(2) Polyurethanes:

The polyurethanes were made by parallel synthesis on a millimolar scale via a typical two-stage poly-addition reaction.

Typically a pre-polymer was prepared by the reaction of polydiol (1 equiv) with diisocyanate (2 equiv) in THF and, where used the chain extender (1 or 2 equiv) was added for a second polymerization step. The library of polymers was tailored by changing the nature and amount of the polydiol, the diisocyanate and the chain extender. The polydiol molar mass was varied from 250 to 2000Da, and the ratio between chain extender and polydiol from 1 to 2. The polymers of the two libraries were characterised by using high-throughput GPC (column PLgel HTS-D 150 * 7.5 mm ID, Polymer Laboratories, 1-methyl-2- pyrrolidinone(NMP)), Hyper DSC (Diamond, Perkin Elmer) and FT-IR (Satellite, Mattson instrument).

Typical Mw results for the polyurethanes prepared were of the order of 40,000 to 210,000 Daltons, however polymers may be prepared with different Mw profiles by appropriate adjustment of the production method as known to those skilled in the art. Evaluation 1

Bacteria culture: Bacteria that express Green Fluorescent Protein (GFP)¹² within the bacteria were employed, allowing ease of detection of bacterial binding on a polymer microarray.

S. typhimurium SL1344¹³ and E. coli W3110¹⁴ transformed with pHC60 (referred to as S. typhimurium-GFP and E. coli-GPP)⁵ were grown overnight with aeration at 37°C or 30°C respectively in Luria-Bertani (LB) broth containing tetracycline (10 ml^"1). Cultures were collected by centrifugation, washed with fresh LB broth and diluted tenfold to a final concentration of approximately (2x10⁸ CFU mL^"1) for microarray binding studies.

Bacterial binding: Either S. typhimurium-GFP or E. co//^'-GFP were added to duplicate polymer microarrays in a four-well plate (NUNC) and incubated overnight (except where stated) at room temperature. Subsequently, the polymer microarray slides were washed with gentle shaking three times with PBS, rinsed in deionised water, and dried with a stream of air. A GeneFrame and coverslip (1.9 x 6.0 cm, AB-0630, Thermo Scientific) was applied to each slide. Polymer microarrays were analysed using a LaVision BioAnalyzer 4F/4S scanner with a FITC filter. Bacterial adhesion was evaluated via integration of the fluorescence intensity after background correction. The average and the standard deviation for sets of four identical polymer features were determined, with the reproducibility between two identical microarrays evaluated by a student t-test. Polymers with p-values < 0.001 and 6 degrees of freedom were considered statistically significant. Fluorescence-based high-content imaging: Imaging was carried out using an automated fluorescent microscope with an XYZ stage running Pathfinder™ (IMSTAR) that allowed the capture of single images for each polymer spot. Bacteria were imaged with both bright-field and Fluorescein channels with a *20 objective. The results are discussed below.

Scanning electron microscopy (SEM): Bacteria on the polymer samples were washed (x2) with 0.1 M cacodylate buffer (pH 7.4) and then fixed with 2.5% (w/v) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h. Samples were post fixed with 1 % (w/v) osmium tetroxide for 1 h at room temperature, dehydrated stepwise with ethanol (50, 70, 90 and 100% (v/v)), critical point dried in C0₂ and gold coated by sputtering. The samples were examined with a Philips XL30CP Scanning Electron Microscope. Coverslip scale-up: Polymers selected for study at a larger scale were spin-coated onto gridded glass coverslips (CELL-VU DRM 800) and incubated with S. typhimurium GFP and imaged via SEM. The number of bacteria in randomly selected sub-squares (four for each coverslip) were counted with Image-Pro Plus 4.5 (©2001 Media Cybernetics). Reproducibility was determined by calculating the average and the standard deviation for the four identical sub-squares.

RESULTS OF EXPERIMENTS - Evaluation 1

Bacterial binding polymers

Tables 4 (poiyacrylate) and 5 (polyurethanes) below show the results from microarray testing of selected polymers, showing strong binding of bacteria. The evaluation of binding of bacteria in the right hand columns is expressed as follows in all the following Tables of results: VV= >50,000 bacteria/mm²; V= >20,000 bacteria/mm²; VX=5,000-20,000 bacteria/mm²; X = <5,000 bacteria/mm²; XX = <1 ,000 bacteria/mm²; ?= not tested yet.

~X indicates a result of from 5,000 - 20,000 bacteria/mm² but sufficiently close to 5,000 bacteria/mm² to be considered potentially category X in view of the expected range of experimental error in testing. Similarly ~V indicates a result of from 5,000 - 20,000 bacteria/mm² but sufficiently close to 20,000 bacteria/mm² to be considered potentially category V in view of the expected range of experimental error in testing.

As applied here a standard glass surface, generally considered a non- (or not very) binding surface for bacteria would fall in the region of 5-10,000 bacteria/mm² for S. typhimurium and 1-5,000 bacteria/mm² for E. coli.

Table 4

Table 5

S. t himurium and E. coli ol urethane bindin anal sis

Six polyacrylate polymers and thirteen polyurethane polymers showed strong binding of S. typhimurium. E. coli affinity was weaker in general, but varied with the particular polymer.

Four of the six high binding polyacrylate polymers (155, 172, 181 and 182) contained the monomer 2-hydroxyethylmethacrylate (HEMA) (see Table 4) and of those four, two (PA181 and 182) contained the monomer 1-vinylimidazole (VI) with monomer ratios: 70/30 and 50/50, respectively (Table ). Of the six high binding polyacrylate polymers five contained hydroxyalkyi groups (monomers HEMA or HBMA) and the other (Pg17) acrylic acid (A-H) in combination with MEMA and DEAEA.

In the case of the polyurethanes, polymer structure analysis of the polyurethanes revealed that the polydiols: polybutylene glycol (PTMG) and polypropylene glycol (PPG) were common in ten of the thirteen "hit" polymers.

Variation in binding for different organisms is also demonstrated. Thus for example, polymer PU222 showed high amounts of binding of S. typhimurium and £. coli, whereas there was a substantial difference between the binding of S. typhimurium and E. co//^' on polymer PU178 (Table 5).

Non-binding and selectively binding polymers

Tables 6 and 7 below show the results of binding tests for 16 selected polyacrylates that exhibit substantial inhibition to S. typhimurium. In Table 6 eleven polyacrylates using amine functionalised GMA are listed and in Table 7 five polyacrylates using amine containing acryiate monomers (DEAEMA 2-(diethylamino)ethyl methacrylate or DEAEA 2-(diethylamino)ethyl acryiate) are listed. The polymer compositions are as defined in Tables 3 above.

Table 6

S. typhimurium and E. co// polyacrylate binding analysis

Functionalisation Amines S. typhimurium E. coii

PA306 Di-n-Butylamine XX X

PA321 cyclohexanemethylamine XX Vx

PA322 cyclohexanemethylamine XX vx

PA323 cyclohexanemethylamine XX VX

PA325 Benzylmethylamine XX VX

PA326 Benzylmethylamine XX XX

PA327 2-(2-Methylaminoethyl) XX X pyridine

PA331 Pyrrole XX v

PA336 N-methylaniline XX X

PA337 N-methylaniline XX v

PA338 N-methylaniline XX v

Table 7

These sixteen polyacrylate polymers showed substantial inhibition of S. typhimurium adhesion, with thirteen containing the monomer methyl methacrylate (MMA), and with eleven of these also containing the monomer glycidyl methacrylate (GMA) that had been derivatised by reaction with a secondary amine.

PA235 and PA236, which are composed of methyl methacrylate (MMA), methacrylic acid (MA-H) and 2-(diethylamino)ethyl methacrylate (DEAEMA), were highly successful in preventing adhesion of both S. typhimurium and E. coli (Table 7).

Polymers PA331 , PA337 and PA338 selectively bound E. coli, but did not bind S. typhimurium. PA337 and PA338 differ only in the molar ratios of the relevant monomers (MMA and GMA): 70/30 (PA337) and 50/50 (PA338) (Tables 3 and 6). The related polymer PA336 (90/10) showed a similar trend, but with slightly less selectivity (Table 6). This indicates the importance of GMA functionalisation with N-methylaniline (MAn) in making this group of polymers selective for E. coli. Reproducibility

Following the initial analysis of the entire library (in duplicate and with eight copies of each polymer), several polymers which resulted in the strongest or poorest binding of S. typhimurium were re-printed and re-examined with each polymer printed in a 5*5 pattern. Of the four good binding polymers examined (PU104, PA155, PU120, PU126), each showed consistent cellular attachment, whilst the four poor binding polymers (PA325, PA422, PA426, PA235) confirmed their "anti-bacterial" binding properties (see Tables 6 and 7).

Impact of time on attachment

It would clearly be advantageous for a polymer to be able to bind bacteria in a rapid time frame. Therefore, to test the rapidity of S. typhimurium binding, an array with the letters 'UK' repeated four times in polymer spots (Figure 1 a) was fabricated using high and low binding polymers (PU104 and PA325, respectively) for different letters. S. typhimurium was incubated on the array for four hours. As can be seen (Fig 1 b fluorescence image), a uniform binding pattern was observed with PU104, with little binding observed with polymer PA325.

Binding morphology

S. typhimurium binding on several selected polymers (PU104, PA155, PU120, PU126, PA325, PA422, PA426 and PA235) was assessed with particular attention paid to the binding characteristics and polymer spot morphology by making use of SEM imaging. Bacteria appeared firmly attached and closely packed on PA155, aligning along their longitudinal axis. Small micro-colonies were observed on the strong-binding polymer surface. In contrast, non-binding polymers (PA325) showed little attachment and very little evidence for early biofilm formation, indicating that such polymers could find use as new materials for antibacterial surface coatings.

Scale-up analysis

PA155 and PA325 were spin-coated onto glass coverslips, which were formed of a central square (1 x 1 mm) subdivided in one hundred squares (100 x 100 pm). These coated coverslips and uncoated coverslips (as a control), were incubated with S. typhimurium-GFP and imaged via SEM. The number of bacteria on randomly selected subsquares on the coverslips were counted to give the number of bacteria per mm². The analysis of binding on both coated and uncoated coverslips confirmed the expected results. S. typhimurium attached onto polymer PA155 with a 7-fold increase in binding compared to an uncoated coverslip, whereas the number of S. typhimurium on the anti-binding polymer PA325 was twenty times less than the glass control. Evaluation 2

Experimental

Bacteria culture: S. enteritidis ⁵ were grown overnight with aeration at 37°C Luria- Bertani (LB) broth. Cultures were collected by centrifugation, washed with fresh LB broth and diluted tenfold to a final concentration of approximately (2x10⁸ CFU mL^"1) for microarray binding studies.

Bacterial binding: S. enteritidis were added to duplicate polymer microarrays in a four- well plate (NUNC) and incubated overnight (except where stated) at room temperature. Subsequently, bacteria on the polymer microarray were stained with DAPI (1 mg/ml) for 20 min, and the polymer microarray slides were washed with gentle shaking three times with PBS, rinsed in deionised water, and dried with a stream of air. A GeneFrame and coverslip (1.9 x 6.0 cm, AB-0630, Thermo Scientific) was applied to each slide. Polymer microarrays were analysed using a LaVision BioAnalyzer 4F/4S scanner with a DAPI filter. Bacterial adhesion was evaluated via integration of the fluorescence intensity after background correction. The average and the standard deviation for sets of three identical polymer features were determined, with the reproducibility between two identical microarrays evaluated by a student t-test. Polymers with p-values < 0.001 and 6 degrees of freedom were considered statistically significant. The results were ranked from the strongest binding polymer to the poorest one within the tested polymers.

Fluorescence-based high-content imaging: Imaging was carried out using an automated fluorescent microscope with an XYZ stage running Pathfinder™ (IMSTAR) that allowed the capture of single images for each polymer spot. Bacteria were imaged with both bright-field and DAPI channels with a *20 objective.

RESULTS OF EXPERIMENTS - Evaluation 2

The results of evaluation 2 are tabulated below (Table 8 for polyacrylates and Table 9 for polyurethanes) using the same indication of binding of bacteria as used in the tables of evaluation 1.

VV= >50,000 bacteria/mm²; V= >20,000 bacteria/mm²; Vx=5,000-20,000 bacteria/mm²; X = <5,000 bacteria/mm²; XX = <1 ,000 bacteria/mm²; ?= not tested yet. ~X indicates a result of from 5,000 - 20,000 bacteria/mm² but sufficiently close to 5,000 bacteria/mm² to be considered potentially category X in view of the expected range of experimental error in testing. Similarly -V indicates a result of from 5,000 - 20,000 bacteria/mm² but sufficiently close to 20,000 bacteria/mm² to be considered potentially category V in view of the expected range of experimental error in testing.

Table 8

continued on next page/ Table 8 (continued)

continued on next page/ Table 8 (continued)

PA for poor PA168 HEMA DMAPMAA - X ~x XX binding of PA230 MMA A-H DEAEMA X XX XX three PA234 -X

MMA MA-H DEAEMA

XX X

strains

PA235 MMA MA-H DEAEMA XX XX X

PA236 MMA MA-H DEAEMA XX XX X

PA242 MEMA A-H DEAEMA XX XX XX

PA475 MEMA DEAEA HEMA XX X X

PA481 MEMA DEAEMA A-H X XX XX

PA493 MEMA DEAEA MA-H X X XX

PA99 XX XX X

MEMA DMAEMA -

Table 9

S. typhimurium, E. coli and S. enteritidis polyacrylate binding analysis

continued on next page/ Table 9 (continued)

PU for PU142 PTMG 250 BICH PG ? ? binding of PU271 PEG 400 MDI DMAP ? ? v

S. D

enteritidis

PU for PU67 PPG 2000 MDI BD X XX ~v selective

binding of

S.

enteritidis

PU for XX selective PU163 PTMG 2000 MDI EG ~v binding of

S.

typhimuri

um and

E. coli

PU for PU16 PEG 2000 MDI none ? ? XX poor PU21 PEG 2000 PDI none ? ? XX binding of PU27 PEG 900 HMDI none ? ? XX

S. PU5 PTMG 2000 HDI none ? ? XX enteritidis PU6 PEG 2000 BICH none ? ? XX

PU71 PEG 2000 PDI BD ? ? XX

PU for PU65 PEG 400 MDI BD ~ X V selective

binding of

S. typh

and S.

enter

PU for PU1 PEG 2000 HDI none ~x X XX poor PU4 PPG 2000 HDI none ~x XX XX binding of

three

strains The headings S typh. and S enter, refer to S. typhimurium and S. enteritidis in Table 9.

One polyacrylate (PA181 ) and twelve polyurethanes showed strong binding to all three strains of bacteria tested. In the binding polyurethanes, some of them showed similar chemical structures, as the examples of PU118 and PU1 19, PU219 and PU222 respectively.

At least eight poiyacryiates and one polyurethane (PU161 ) showed selective binding for E. coli rather than for the two Salmonella strains. As showed in Table 8, PA336, PA337, and PA338 have the same polymer backbone and the same amine functionalisation, but PA336 has less bacterial selectivity that may be due to less An functionalisation on the GMA ( MA/G A 90/10).

Five poiyacryiates and two polyurethanes showed strong binding to S. enteritidis, (PA204, PA210, PA213, PA36, PA384, PU142 and PU271 ), although selectivity remains to be determined.

Ten poiyacryiates and potentially two polyurethanes showed poor binding for all three strains. In particular, PA234, PA235 and PA236, which have the same combination of monomers but varied monomer ratios, were highly effective for bacteria repellence.

Evaluation 3

The focus of this evaluation was on major bacterial pathogens associated with food- borne infection, ventillator associated pneumonia or cardiovascular implants.

Experimental

Bacteria and growth conditions: The organisms used in this evaluation were poultry isolates of Campylobacter jejuni, or clinical isolates of Clostridium difficile and perfringens, Staphylococcus aureus, Enterobacter (unidentified species), Streptococcus viridans (group E), Enterococcus faecalis or endotracheal tube isolates from intensive care unit patients, Klebsiella pneumoniae, and Staphylococcus saprophytics..

Organisms were grown at 37°C, overnight with aeration (Staphylococcus aureus, Klebsiella pneumoniae, Staphylococcus saprophytics and Enterobacter) or statically and under microaerophilic {Campylobacter (grown for 48 hours), Enterococcus and Streptococcus viridans; 10% C0₂ and 5% O2) or anaerobic conditions (Clostridium spp.).

Organisms were initially cultured in Luria-Bertani (LB) broth (Klebsiella,enterobacter and Staphylococcus spp.), Brain Heart Infusion broth (Enterococcus, Streptococcus, Clostridium spp.), or Brucella broth supplemented with iron (ii) sulphate, sodium pyruvate and sodium metabisulphite) and antibiotics vancomycin and trimethoprim (final concentrations: 2500 units/I and 5mg/l respectively); Campylobacter spp.).

Cultures were then processed as in evaluation 2, except where stated below. Bacteria were harvested by centrifugation, washed with fresh medium and diluted tenfold for microarray binding studies. In addition to the individual organisms shown in figures 2 and 3, two clinical cocktails were examined with the arrays.

Clinical cocktail 1 was based on endotracheal tube associated organisms. The washed cultures of Klebsiella pneumoniae, Enterobacter, Staphylococcus saprophytics, Staphylococcus aureus were therefore mixed equally prior to addition to the microarray and subsequent analysis as in Evaluation 2.

Clinical cocktail 2 was based on major organisms associated with implant-related infectious endocarditis and comprised Staphylococcus aureus, Streptococcus viridans (group E), and Enterococcus faecalis. In this case, BHI medium was used for washing and samples were mixed equally prior to incubation with the arrays. Array incubation was then undertaken as in evaluation 2 with the exception that for clinical cocktail 2, arrays were incubated vertically, as opposed to horizontally with mild rotational agitation (approximately 50 r.p.m.).

Results

The results of evaluation 3 are tabulated in figures 2 and 3 (Figure 2 for polyacrylates and Figure 3 for polyurethanes) using the same indication of binding of bacteria as used in the tables of evaluations 1 and 2. H= >50,000 bacteria/mm²; V= >20,000 bacteria/mm²; VX=5,000-20,000 bacteria/mm²; X = <5,000 bacteria/mm²; XX = <1 ,000 bacteria/mm²; ?= not tested yet.

~X indicates a result of from 5,000 - 20,000 bacteria/mm² but sufficiently close to 5,000 bacteria/mm² to be considered potentially category X in view of the expected range of experimental error in testing. Similarly -V indicates a result of from 5,000 - 20,000 bacteria/mm² but sufficiently close to 20,000 bacteria/mm² to be considered potentially category V in view of the expected range of experimental error in testing. Figures 2 and 3 show results of testing against eight strains of bacteria and two mixtures of Clinical isolates.

Bacterial Binding polymers

Polyacrylates

For Campylobacter, no polyacrylates with particularly strong binding for both strains were observed, although PA60 and 181 should a reasonable degree of binding for strain 2. However this also extended to other bacteria. For S. viridans, no unique binding polyacrylates were detected but reasonable to strong binding was observed for polymers such as PA181 , PA182 and PA416 (as with a range of other organisms thereby indicating broad spectrum activity). The latter polymer was also found to bind well to Clostridium perfringens (indicating activity towards a range of Gram positive and negative organisms). The clinical mixtures differed in their binding pattern with mixture 2 showing some degree of binding to polymers such as PA323, PA324 and PA327. PA337 and 338 and to a lesser extent, PA316 and PA504, were found to be particularly good at selectively binding E. coli. PA 163 and 485 also showed selective binding for S. typhimurium whilst PA324 show preference for binding S. enteritidis. Some degree of selective binding by Gram positive organisms (S. viridans and C. perfringens) was observed with PA426 and 493. Polyurethanes

A degree of selective binding was observed for C. jejuni 1 for PU67 whilst C. jejuni isolate 2 exibited stronger binding with a number of polymers (e.g. PU1 18, PU119, PU208). The Gram positive organism S. viridans showed good binding for polymers PU92, PU1 18 and PU126, which was shared with several Gram negative organisms. In general, the Clostridium strains exhibited moderate to very poor binding (repellence) by the polyurethane polymers. Interestingly though, PU118 and PU219 showed good binding for bacteria in the clinical mixture 2 and this was shared by a number of the other organisms (particularly for PU118). Bacterial repellant polymers

Polyacrylates.

Overall the polyacrylates listed in Figure 2 exhibited repellent properties (e.g. PA168, 306, 475 and 481 ), reinforcing the value of these examples as broad range repellents for Gram positive and negative organisms and including the clinical isolates. Whilst some degree of binding was observed for the Gram positive organisms S. mutans and C. perfringens, the Gram negative organisms (Salmonella, E. coli and Campylobacter) exhibited poor binding with PA327, 426 and 493.

Polyurethanes

PU1 showed good overall repellent properties for all organisms examined, as did PU161 (with the exception of E. coli). A degree of discrimination was observed between Gram positive and negative organisms for PU1 16, PU120, PU159, and PU220, with the latter organisms showing binding.

References

1 H. Morisaki and H. Tabuchi, Colloids Surf. B, 2009, 74, 51-55.

2 D. G. Anderson, S. Levenberg and R. Langer, Nat. Biotech., 2004, 22, 863-866.

3 D. G. Anderson, D. Putnam, E. B. Lavik, T. A. Mahmood and R. Langer, Biomaterials, 2005, 26, 4892-4897.

4 A. L. Hook, D. G. Anderson, R. Langer, P. Williams, M. C. Davies and M. R. Alexander, Biomaterials, 31 , 187-198.

5 A. L. Hook, H. Thissen and N. H. Voelcker, Biomacromolecules, 2009, 10, 573- 579.

6 S. E. How, B. Yingyongnarongkul, M. A. Fara, J. J. Diaz-Mochon, S. Mittoo, M. Bradley, Comb. Chem. High Throughput Screen 2004, 7, 423-430.

7 G. Tourniaire, J. Collins, S. Campbell, H. Mizomoto, S. Ogawa, J. F. Thaburet, M. Bradley, Chem. Commun., 2006, 20, 2118-2120.

8 R. Zhang, A. Liberski, F. Khan, J. J. Diaz-Mochon and M Bradley, Chem. Commun., 2008, 11 , 1317-1319.

9 (1 ) S. Pernagallo, A. Unciti-Broceta, J. J. Diaz-Mochon and M. Bradley, Biomed. Mater., 2008, 3, 034112. (2) A. Liberski, R. Zhang and M. Bradley, Chem. Commun., 2009, 3, 334-336.

10 F. Khan, R. S. Tare, J. M. Kanczler, R. O. C. Oreffo and M. Bradley, Biomaterials, 2010, 31 , 2216-2228.

11 (1 ) S. Pernagallo, J. J. Diaz-Mochon, M. Bradley, 2009, Lab Chip, 9, 397-403. (2) H. Mizomoto, PhD thesis, University of Southampton, Southampton (2004). (3) J. F. O. Thaburet, H. Mizomoto, M. Bradley, Macromol. Rapid Commun., 2004, 25, 366-370. (4) A. Unciti-Broceta, J. J. Diaz-Mochon, H. Mizomoto and M. Bradley, J. Comb. Chem., 2008, 10, 179-184.

12 R. Heim, A. B. Cubitt and R. Y. Tsien, Nature, 1995, 373, 663-664.

13 S. K. Hoiseth and B. A. Stocker, Nature, 1981 , 291 , 238-239.

14 M. Riley, T. Abe, M. Arnaud, M. Berlyn, F. Blattner, R. Chaudhuri and J. D. Glasner, Nucleic Acids Res., 2006, 34, 1-9.

15 E. F. Boyd, F. S. Wang, P. Beltran, S. A. Plock, K. Nelson, R. K. Selander, J. Gen Microbiol 1993, 139, 1125-1132.

Claims

1. Use of a polymer to prevent binding of a microorganism to a substrate:

wherein the polymer is a polyacrylate polymer, that is non-binding or selectively binding to microorganisms, said acrylate polymer having a methacrylate or acrylate containing polymer backbone with pendant tertiary amine groups of the structure;

wherein X is a linker group bonding to the polymer backbone and is selected from the group consisting of substituted or unsubstituted alkylene that may be cyclic and may be unsaturated ; substituted or unsubstituted aryl or heteroaryl; and

R' and R" are each independently selected from:

alkyl groups that may be saturated or unsaturated and may be substituted; aryl or heteroaryl groups that may be substituted; or

R' and R" are fused to form a ring that may be saturated or unsaturated, may contain heteroatoms and may be substituted.

2. The use according to claim 1 wherein the linker groups X are bonded to the polymer backbone via an ester or amide function of a methacrylate or acrylate monomer used in the preparation of the polymer.

3. The use according to claim 2 wherein the pendant tertiary amine groups have the structure: or or

,N.

R" R' N

N-R'

R" R'

R"

4. The use according to claim 3 wherein the polymer comprises or consists of EMA or MMA; and

a second monomer that either: contains the tertiary amine function; or that has been reacted with a secondary amine to give the tertiary amine function.

5. The use according to any preceding claim wherein between 5% to 60%, of the monomers forming the polymer backbone are functionalised with the pendant tertiary amine groups.

6. The use according to any preceding claim wherein the polymer is selected from the group consisting of polymers prepared by polymerisation of one of the following groups of two or three monomers:

Ratio (mol)

Monomer 1 Monomer 2 Monomer 3

mon 1 mon 2 mon 3 EMA DMAEMA - 90 10 -

HEMA DMAPMAA - 90 10 -

MMA A-H DEAEMA 70 10 20

MMA MA-H DEAEMA 70 20 10

MMA MA-H DEAEMA 70 15 15

MMA MA-H DEAEMA 70 10 20

MEMA A-H DEAEMA 70 10 20

MMA GMA DnBA^* 90 10 -

MMA GMA cHMA^* 90 10

MMA GMA cHMA* 70 30 -

MMA GMA cHMA* 50 50 -

MMA GMA BnMA^* 70 30 -

MMA GMA BnMA* 50 50 -

MMA GMA MAEPy* 90 10 -

MEMA DEAEMA BMA 40 30 30

MEMA DEAEA BMA 40 30 30

MEMA DEAEA HEMA 60 10 30

MEMA DEAEMA A-H 80 10 10

MEMA DEAEA MA-H 80 10 10

MEMA DEAEA St 60 10 30

MEMA DMVBA - 50 50 -

BMA DEAEA - 50 50 -

BMA DMAEA - 50 50 -

MEMA GMA DnBA^* 70 30 -

MEA PAA - 50 50 - HBMA DEAEA - 90 10 - MA GMA BnMA^* 90 10 - wherein, when monomer 2 is GMA, sufficient monomer 3 is reacted with the epoxide function of the GMA to convert all the epoxide groups to pendant tertiary amine groups on the polymer.

7. The use according to claim 6 wherein the polymer is selected from the group consisting of polymers prepared by polymerisation of one of the following groups of two or three monomers:

wherein, when monomer 2 is GMA, sufficient monomer 3 is reacted with the epoxide function of the GMA to convert all the epoxide groups to pendant tertiary amine groups on the polymer.

8. The use according to any preceding claim wherein the microorganism is a bacterium.

9. The use according to claim 8 wherein the microorganism is a bacterium selected from the group of species of the genus: Salmonella, Escherichia (coli), Staphylococcus, Clostridium, Campylobacter, Clostridium, Streptococcus, Klebsiella, Staphylococcus, Enterobacter, and Enterococcus.

10. The use according to claim 9 wherein the microorganism is a bacterium selected from the group of: E. coli , S. typ imurium, S. enteritidis, Campylobacter jejuni, Clostridium perfringens, Clostridium difficile, Streptococcus viridans, Klebsiella pneumoniae, Staphylococcus saprophytics, Staphylococcus aureus ,Enterobacter, Enterococcus faecalis, and Streptococcus mutans.

11. The use according to any preceding claim wherein the substrate is provided with a coating, the coating comprising, consisting of or consisting essentially of the polymer.

12. The use according to any one of claims 1 to 10 wherein the substrate comprises, consists of, or consists essentially of the polymer.

13. A coating for a substrate comprising, consisting of or consisting essentially of a polyacrylate polymer, that is non-binding or selectively binding to microorganisms, said acrylate polymer having a methacrylate or acrylate containing polymer backbone with pendant tertiary amine groups of the structure;

wherein X is a linker group bonding to the polymer backbone and is selected from the group consisting of substituted or unsubstituted alkylene that may be cyclic and may be unsaturated; substituted or unsubstituted aryl or heteroaryl; and

R' and R" are each independently selected from:

14. A medical device, comprising, consisting of, consisting essentially of or coated with a polyacrylate polymer, that is non-binding or selectively binding to microorganisms, said acrylate polymer having a methacrylate or acrylate containing polymer backbone with pendant tertiary amine groups of the structure;

R' and R" are each independently selected from:

15. A medical device according to claim 14, wherein the medical device is an implant.

16. A medical device according to claim 14 selected from the group consisting of tubes, stents, catheters, replacement joints, replacement joint components, wound dressings, surgical gauze, tooth coatings or implants, endoscopes, surgical tools and contact lenses.

17. An article selected from the group consisting of a tool, a work surface, a platform for molecular or cellular fabrication, a component of a water supply system a face mask, a protective body suit, a toothpaste, a mouthwash and a paint;

wherein said article comprises, consists of, consists essentially of, or is coated with a polyacrylate polymer, that is non-binding or selectively binding to microorganisms, said acrylate polymer having a methacrylate or acrylate containing polymer backbone with pendant tertiary amine groups of the structure;

X

R"^ ^R'

R' and R" are each independently selected from:

18. Use of a polymer to bind a microorganism to a substrate wherein the polymer is a polyacrylate polymer that is binding to said microorganism;

wherein said polyacrylate polymer comprises hydroxyalkyi or methoxyalkyl functional groups.

19. The use according to claim 18 wherein the hydroxyalkyi or methoxyalkyl functional groups have an alkyl function of from 1 to 10 carbon atoms.

20. The use according to claim 18 wherein the hydroxyalkyi or methoxyalkyl functional groups are selected from the group consisting of hydroxyethyl, hydroxybutyl and methoxyethyl.

21. The use according to any one of claims 18 to 20 wherein the polyacrylate polymer comprises hydroxyalkyi functional groups derived from a hydroxyalkyi methacrylate monomer and said polyacrylate polymer is prepared from a monomer mixture having a molar ratio range of from 95:5 to 45:55 hydroxyalkyi methacrylate monomer to other monomers employed in the mixture.

22. The use according to claim 21 wherein the hydroxyalkyi methacrylate monomer is copolymerised with at least one of: DEAA [diethylacrylamide], DMAEMA [2- (dimethylamino)ethyl methacrylate], BACOEA (2-[[(butylamino)carbonyl]oxy]ethyl acrylate) and VI [1-vinylimidazole].

23. Use of a polymer to bind a microorganism to a substrate wherein the polymer is a polyurethane polymer that is binding to said microorganism;

wherein said polyurethane polymer is formed by polymerising a polydiol with a di-isocyanate and optionally with an extender molecule.

24. The use according to claim 23 wherein said diol is selected from the group consisting of:

PPG: poly(propylene glycol);

PTMG: poly(tetramethylene glycol) also known as poly(butylene glycol); PHNGAD: poly[1 ,6-hexanediol/neopentyl glycol/diethylene glycol-alt-(adipic acid)]diol; PHNAD: poly[1 ,6-hexanediol/neopentyl glycol-alt-(adipic acid)]diol; and PEG: poly(ethylene glycol).

25. The use according to claim 23 or claim 25 wherein said di-isocyanate is selected from the group consisting of:

HDI: 1 ,6-diisocyanohexane; MDI: 4,4'-methylenebis(phenylisocyanate);

TDI: 4-methyl-1 ,3-phenylene diisocyanate; PDI: 1 ,4-diisocyanobenzene;

HMDI: 4,4'-methylenebis(cyclohexylisocyanate); and

BICH: 1 ,3-bis(isocynanatomethyl)cyclohexane.

26. The use according to any one of claims 23 to 25 wherein an extender molecule is employed and is selected from the group consisting of:

BD: 1 ,4-butanediol; EG: ethylene glycol; PG: propylene glycol;

ED: ethylene diamine; OFHD: 2,2,3,3,4,4,5,5-octafluoro-1 ,6-hexanediol;

DEAPD: 3-diethylamino-1 ,2-propanediol; DMAPD: 3-dimethylamino-1 ,2- propanediol; and NMPD: 2-nitro-2-methyl-1 ,3-propanediol.

27. The use according to any one of claims 18 to 26 wherein the microorganism is a bacterium.

28. The use according to any one of claims 18 to 27 wherein the substrate is provided with a coating, the coating comprising, consisting of or consisting essentially of the polymer.

29. The use according to any one of claims 18 to 27 wherein the substrate comprises, consists of, or consists essentially of the polymer.

30. An article selected from the group consisting of: a swab, a wound dressing, a slow release structure, a cleaning cloth, a cleaning liquid and filter media; said article comprising, consisting of or consisting essentially of a polymer for use in binding a microorganism to a substrate according to any one of claims 16 to 25.

31. A slow release structure according to claim 30 for use in therapy.

32. Use of a polymer to prevent binding of a microorganism to a substrate: wherein the polymer is a polyacrylate polymer, that is non-binding or selectively binding to microorganisms, and selected from the group consisting of polymers prepared by polymerisation of one of the following groups of two monomers:

33. Use of a polymer to prevent binding of a microorganism to a substrate:

wherein the polymer is a polyurethane polymer, that is non-binding or selectively binding to microorganisms, wherein said polyurethane polymer is formed by polymerising a polydiol with a di-isocyanate and optionally with an extender molecule; and wherein the polydiol is a polyethylene glycol or the extender molecule is ethylene glycol.

34. Use of a polymer to prevent binding of a microorganism to a substrate:

wherein the polymer is a polyurethane polymer, that is non-binding or selectively binding to microorganisms, wherein said polyurethane polymer is formed by polymerisation of one of the following groups of polydiol, diidocyanate and extender molecules:

Polymer Composition

Ratio (mol)

Polydiol Mn Dis. Ext.

Diol Dis Ext

PEG 400 MDI BD 25 52 23

PTMG 650 MDI EG 25 52 23

PEG 2000 HDI none 48.5 51.5 0

PPG 2000 HDI none 48.5 51.5 0

PPG 425 BICH BD 48.5 51.5 0

PPG 425 BICH DEAPD 25 52 23

PTMG 250 MDI BD 25 52 23

PHNAD 900 MDI DMAPD 25 52 23