The present invention relates to a method for producing a silver-containing polymer and, in particular, a silver-containing polyurethane. Such polymers have antimicrobial properties. The present invention also relates to the use of such polyurethanes in the manufacture of medical devices and, in particular, implantable medical devices.

Microbial contamination is a substantial problem for medical devices and healthcare products. In particular, implantable medical devices are vulnerable to microbial colonization and biofilm formation. Devices such as urethral catheters, sphincter pressure sensors or devices used in central venous lines are all recognized as being susceptible to bacterial colonization and infection. Complications which can result as a consequence of such infections include extended tissue damage, device failure and the spread of the infection to the other areas. Previous studies have shown that, in order to develop an antibacterial or infection-resistant material, a broad antibacterial activity achieved by reducing or preventing bacterial adherence and avoiding biofilm formation.

Various approaches have been proposed and tested to extend protection of implantable materials against microbial colonization and biofilm formation. For instance, surfaces of medical devices have been modified, either by using surface modifying agents or by impregnation with antiseptics and antibiotics, to reduce the adherence of bacterial microorganisms. However, these methods have only been partially successful and providing medical devices with long-term antimicrobial properties remains a challenge. Silver ions are known to have antimicrobial activity due to their ability to disrupt bacterial protein synthesis. For this reason, silver has been incorporated into polymers that are intended for medical applications, such as polyurethanes. Conventionally, silver is incorporated by depositing metallic silver onto the surface of the polymer. More recently, however, polyurethane polymers containing co-ordinated silver have been produced. For example, Acta Biomaterialia 6 (2010) 3482 - 3490 describes a process in which a sodium salt of a carboxylated polyurethane silver nitrate to produce a silver-containing polyurethane. The carboxylated polyurethane, however, contains one carboxylate group per repetitive unit along its chain. This can have a significant effect on, for example, the mechanical properties and aqueous stability of the polymer. It is among the objects of embodiments of the invention to provide an alternative or improved method of incorporating silver into a polymer, such as polyurethane. According to a first aspect of the present invention, there is provided a method of producing a silver-containing polymer, said method comprising:

grafting a silver compound to a polymer, whereby the silver compound is grafted to the polymer via a urethane or urea linkage.

The method of the present invention may be used to incorporate silver into any suitable polymer. For instance, homopolymers and copolymers may be used. Suitable polymers may advantageously contain urethane or urea linkages. Examples include polyurethanes and, in particular, polyetherurethanes. Alternatively or additionally, the polymers may be formed from a precursor polymer containing groups, such as hydroxyl, ester, ether or amine groups, that can be reacted with isocyanate groups to form urethane or urea linkages. Examples of suitable precursor polymersinclude polyethers, silicone (e.g. polydimethylsiloxane), polymethylmethacrylate (PMMA), polycarbonate, polyether, polyamide and polyester. Polyethers are preferred. In the method of the present invention, the polymer or precursor polymer may be formed by performing a polymerisation reaction to form a polymer chain, and reacting the polymer chain with the silver compound. The polymerisation reaction may be carried out by polymerising monomers or pre -polymers or oligomers together in a homo- or co-polymerisation reaction. When polymerised, these building blocks, referred to in this patent specification as "monomeric units" are linked or bonded together to form the polymer chain. Any suitable monomeric units may be used, such as ethers, esters, alcohols (e.g. diols), dimethylchlorosilane, amides, polyethers, polyesters, polyols and isocyanates. Hydroxyl and amine terminated polyethers, such as hydroxyl and amine terminated polyethers, polyesters, polycarbonates and polydimethylsiloxane may be used. A suitable ester is the methyl ester of methacrylic acid, which, when polymerised, produces a polymer chain of

polymethylmethacrylate (PMMA). Other examples of monomeric units include bisphenol A and phosgene, which may be polymerised to form polycarbonate chains.

In a preferred embodiment, the polymerisation reaction takes place between a polyol (e.g. an oligomeric diol) and an isocyanate, such as a diisocyanate or polyisocyanate, to produce a polyurethane. Specific examples of suitable diisocyanates include methylene diphenyl diisocyante (MDI), toluene diisocyanate (TDI), hexamethylene diisocyanate (HDI) and isophorone diisocyante (IPDI). Preferably, methylene diphenyl diisocyante (MDI) is used. Suitable polyols include those with molecular weights of at least 1000 g mol ¹ , preferably at least 1400g mol ¹ , more preferably at least 1500 g mol ^"1" , for example, from 1500 to 2000 g mol ^"1 . Suitable polyols include polyether and polyester polyols. An example is polytetramethylene oxide, polyhexamethylene oxide and polydimethylsiloxane. The molar ratio of polyol to isocyanate (e.g. diisocyanate) may be about 1 - 20 : 20 - 1; preferably about 1:2. The monomeric units may be polymerised under any suitable conditions. Suitable polymerisation reaction temperatures range from 40 to 150 degrees, preferably 50 to 120 degrees C. In a preferred embodiment, solution polymerisation may be used.

The polymer chain formed in the polymerisation step may be reacted with a chain extender to form an extended polymer chain. Suitable chain extenders include diols and diamines. Such chain extenders typically have molecular weights of below 200 gmol ¹ , preferably below 150 gmol ¹ . Such chain extenders (e.g. diols and/or amines) may also contain fewer than 30 carbon atoms, preferably, fewer than 10 carbon atoms, for example, 2 to 4 carbon atoms. Specific examples of chain extenders include ethylene glycol, 1 ,4-butanediol, 1,6-hexanediol, cyclohexane dimethanol and hydroquinone bis (2-hydroxyethyl)ether. In a preferred embodiment, 1 ,4-butanediol is used. Where a polyol (e.g. polytetramethylene oxide), diisocyanate (e.g. methylene diphenyl diisocyanate) and chain extender (e.g. 1,4-butanediol) are used, the ratio of polyol to diisocyanate to chain extender may be 1— 10: 1— 10: 1 - 10, for example, 1 - 1.1 : 2 - 2.1 : 1 - 1.1 or 1 - 1.05 : 2 - 2.1 : 1 - 1.1. A stoichiometric ratio of 1 :2:1 may be used. Preferably, however, an excess of up to 10% of diisocyanate, up to 10% diol is used and/or up to 10%> chain extender is used.

The unextended or (preferably) extended polymeric chain may be reacted with the silver compound. Advantageously, this reaction grafts the silver compound to the preferably extended polymeric chain. As a result, the silver compound is bonded to the extended polymeric chain by a chemical bond, typically a covalent bond. Any suitable silver compound may be used, including, for example, a silver salt of an organic acid. Preferably, the silver salt is an amine, amide-, cyanide- or hydroxyl-containing silver salt. In other words, the silver compound is preferably a silver salt having an anion (preferably, an organic anion) comprising at least one of a hydroxyl, amide, amine and cyanide group._Specific examples of suitable silver compounds include silver lactate, silver sulfadiazine and silver cyanide. Preferably, silver lactate or silver sulfadiazine is used. Such compounds may be bonded to the unextended or extended polymer chain by a urethane or urea linkage.

Without wishing to be bound by any theory, it is believed that the silver compound serves to quench the reaction. Accordingly, the addition of the silver compound can be used to terminate the polymerisation reaction. By controlling the point at which the silver compound is added to the reaction, therefore, it is possible to control the chain length and, hence, molecular weight of the polymer produced. Accordingly, as well as providing antimicrobial properties, the physical properties of the resulting polymer may be controlled by the addition of the silver compound. As the silver compound quenches the polymerisation reaction, the silver compound is typically grafted to one or both ends of the polymeric chain. In a preferred embodiment, the silver compound is bonded to both ends of the polymer chain, such that the polymer chain is end-capped. Advantageously, the mechanical properties, such as tensile strength, modulus and/or elongation at break, of the polymer chain may remain substantially uncompromised. In one embodiment, the tensile strength of the silver-containing polymer is at least 80%, preferably at least 85%, more preferably at least 90% of that of a corresponding polymer formed in the absence of the grafting step c). Similarly, the modulus and/or elongation at break may be at least 80%, preferably at least 85%, more preferably at least 90% of that of a corresponding polymer formed in the absence of the grafting step c).

In a preferred embodiment, the method of the present invention comprises:

reacting at least one diisocyanate, and at least one polyol to form a prepolymer,

reacting the prepolymer with at least one chain extender to form an extended polymer chain, and

reacting the extended polymer chain with the silver compound.

In one embodiment, the diisocyanate, polyol and chain extender may be reacted together, for example, in a "one-pot" reaction and the silver compound subsequently added to quench the reaction. Preferably, however, the diisocyanate and polyol are reacted together in an initial reaction step to produce a pre-polymer. The pre -polymer is then reacted with the chain extender to form an extended polymer chain before the reaction is quenched by the addition of the silver compound..

Advantageously, by forming a pre-polymer in an initial reaction step, more ordered polymers can be formed.

The chain extender and silver compound may be added to the prepolymer at the same time or separately. Preferably, the chain extender is added to the prepolymer before the addition of the silver compound. As mentioned above, the silver compound quenches or terminates polymerisation. The resulting polymer may be end-capped with the silver compound at one or both ends. Preferably, both ends of the polymer are end-capped with silver.

Suitable diisocyanates may be described above. Examples may be represented in general by the formula: OCNR ₂ NCO wherein R ₂ is an alkylene, cycloalkylene, arylene, substituted-alkylene, substituted-cycloalkylene, substituted arylene or combinations thereof. They can include alicyclic, aliphatic and aromatic diisocyanates. The useful aliphatic and alicyclic diisocyanates include: 1 ,4-cyclohexane

bis(methyleneisocyanate); dicyclohexyl methane 4,4'-diisocyanate; 1 ,4-cyclohexyl diisocyanate; hexamethylene diisocyanate; 1 ,6-diisocyanato-2,2,4,4-tetramethylhexane; 1 ,6-diisocyanato-2,4,4- trimethylhexane; isophorone diisocyanate.

The useful aromatic diisocyanates include napthalene-l,5-diisocyanate, diphenylmethane-4,4'- diisocyanate, toluene diisocyanate, p-phenylene diisocyanate, dibenzyl diisocyanate, diphenyl ether diisocyanate, m- and p-tetramethylxylene diisocyanate, and the like, such as are included in the general formula

OCN-Ar-Y-Ar-NCO wherein Ar is cyclic, i.e. an arylene or alicyclic radical, and Y may be a carbon-to-carbon valence bond, an alkylene radical containing 1 to 5 carbon atoms, oxygen, sulfur, sulfoxide or sulfone.

Suitable polyols are discussed above. The polyol is preferably an oligomeric diol, such as a diol of a polyester, polyether, silicone diol or a combination thereof, of the formula: HOR ₄ OH, wherein R4 is a polyether, polyester, polycarbonate or polydimethylsiloxane having a molecular weight of about 400 to 40,000. Preferably, the molecular weight, M _n , is 1000 to 5000, for example, 1000 to 2000.

Representative polyether glycol reactants, also termed poly(alkylene oxides), are essentially linear hydroxyl containing compounds having ether linkages. The molecular weights preferably vary between about 600 to 4,000. Examples of polyether glycols include hydroxyl terminated

poly(propylene oxide), hydroxyl terminated poly(tetramethylene oxide), hydroxyl terminated poly(trimethylene oxide), hydroxyl terminated poly(hexamethylene oxide), hydroxyl terminated poly(ethylene oxide), and the like, of the formula HO[(CH ₂ ) _n O _x l H wherein n is an integer from 2 to 6 and x is an integer from 5 to 600, and substituted types such as hydroxyl terminated poly(l,2- propylene oxides), hydroxyl terminated poly(l,2-butylene oxide), tetrahydrofuran and ethylene oxide copolyethers, and the like.

Representative polyester glycol reactants include linear polyesters having molecular weights between about 400 and 10,000, and preferably about 1,000 to 4,000. The polyesters utilized include those prepared by the polymerization of esters of aliphatic dicarboxylic acids including, for example, adipic, succinic, pimelic, suberic, azelaic, sebacic and the like or their anhydrides. Aromatic dicarboxylic acids or their anhydrides or mixtures of aliphatic and aromatic dicarboxylic acids or their anhydrides may be used. Useful acids include aliphatic dicarboxylic acids of the formula HOOC-R5 -COOH where R ₅ is an alkylene radical containing 1 to 10 carbon atoms, preferably 4 to 6 carbon atoms. The phthalic acids and their anhydrides are also useful. The glycols used in the preparation of the polyesters by reaction with the dicarboxylic acids are normally aliphatic diols containing between 2 and 10 carbon atoms, usually 2 to 6 carbon atoms, such as ethylene glycol propylene glycol, butanediol, hexamethylene diol, decamethylene diol, 2-ethylhexanediol, 1,6-neopentyl diol and the like. Representative polyester glycols may also include materials such as polycaprolactone diols. Another oligomeric diol which may be used in the present invention is a silicone diol.

Suitable chain extenders are known in the art. As discussed above, suitable chain extenders include diols and diamines. Such chain extenders typically have molecular weights of below 200 gmol ¹ , preferably below 150 gmol ¹ . Such chain extenders (e.g. diols and/or amines) may also contain fewer than 30 carbon atoms, preferably, fewer than 10 carbon atoms, for example, 2 to 4 carbon atoms. Specific examples of chain extenders include ethylene glycol, 1 ,4-butanediol, 1,6- hexanediol, cyclohexane dimethanol and hydroquinone bis (2-hydroxyethyl)ether. In a preferred embodiment, 1 ,4-butanediol is used.

The molar ratio of the polyol to disocyanate to chain extender may be 0.5 - 1.5: 1.5 - 2.5: 0.5 - 1.5, preferably about 1 :2: 1. The molar ratio of the polyol to disocyanate to chain extender to silver compound may be 0.5 - 1.5: 1.5 - 2.5: 0.5 - 1.5: 0.05 - 1, preferably 0.5 - 1.5: 1.5 - 2.5: 0.5 - 1.5: 0.1 - 0.5, for example, 1 :2: 1 :0.2.

The polymerisation may be a solution polymerisation reaction or melt polymerisation reaction. The reaction may be carried out in an inert atmosphere, for example, of nitrogen gas.

Where a solution polymerisation reaction is used, the reaction may also be carried out using any suitable solvent. Suitable solvents are known in the art. In one embodiment, dimethylformamide and/or dimethylsulfoxide are used, for example, in a 1 :1 ratio.

Suitable reaction temperatures are known in the art and range from, for example, 40 to 150 degrees C. In one embodiment, the prepolymer is formed at temperatures from 50 to 80 degrees C. The reaction temperature may then be increased, for example, to 110 to 130 degrees C following the addition of the chain extender and the silver compound. The silver-containing polymer may have an average molecular weight of at least 20,000, preferably at least 50,000, more preferably at least 80,000, yet more preferably at least 100,000. In a preferred embodiment, the silver-containing polymer has an average molecular weight of 80,000 to 200,000. Where the silver-containing polymer has a molecular weight that is lower than desired (e.g. if it is produced from an unextended polymer chain), it may be mixed with a polymer having a higher molecular weight to produce a silver-containing polymer composition having a desired molecular weight.

The silver-containing polymer or silver-containing polymer composition may have a tensile strength of 10 to 50 Mpa , preferably 15 to 30 MPa. The silver-containing polymer or silver- containing polymer composition may have a modulus of 5 to 30 MPa, preferably 10 to 20 MPa. The silver-containing polymer or silver-containing polymer composition may have an elongation at break of 200 to 1000% , preferably 400 to 800%.

The silver content of the silver-containing polymer or silver-containing polymer composition may be 0.01 - 5 weight %>, preferably 0.05 - 2 weight %>, and more preferably 0.01 - 1 weight %>. This silver content provides the polymer with antimicrobial properties. For example, the polymer may be resistant to a range of bacteria, including Staphylococcus bacteria, such as S. aureus.

Advantageously, this antimicrobial activity is long-lasting, as the silver is chemically bonded to the polymer chain. This is believed to reduce or eliminate the risk of excessive amounts of silver leaching out of the polymer with time and/or use.

According to a second aspect of the present invention, there is provided a silver-containing polymer having a polymer backbone and a silver compound that is chemically grafted to the polymer backbone via a urea or urethane linkage.

The polymer may be a polyurethane. Preferably, the polyurethane polymer is a

polyetherurethane polymer having a polyetherurethane backbone that is capped at one or both ends with a silver compound. In one embodiment, the polyurethane polymer (e.g. polyetherurethane polymer) is capped at both ends with a silver compound. Specific examples of such polymers include:

Polyetherurethane-silver lactate

Polyetherurethane-silver sulfadiazine

Any suitable values of n and m may be employed. For example, n and m may each be an integer of 1 or more, preferably 2 or more. In one embodiment, n may be 2 to 1000. m may be at least 2, for example, 10 to 1000. The values of n and m may be controlled according to methods known in the art to achieve a polymer with the desired molecular weight (see above). For example, n and/or m may be controlled such that the resulting polymer has an average molecular weight of at least 20,000, preferably at least 50,000, more preferably at least 80,000, yet more preferably at least 100,000. In a preferred embodiment, the silver-containing polymer has an average molecular weight of 80,000 to 200,000.

The polymer (e.g. polyurethane polymer) may contain silver in an amount of 0.01 - 5 weight %, preferably 0.05 - 2 weight %, and more preferably 0.1 - 1 weight %. This silver content provides the polymer with antimicrobial properties. For example, the polymer may be resistant to a range of bacteria, including Staphylococcus bacteria, such as S. aureus. Advantageously, this antimicrobial activity is long-lasting, as the silver is chemically bonded to the polymer chain. This is believed to reduce or eliminate the risk of excessive amounts of silver leaching out of the polymer with time and/or use.

The polymer (e.g. polyurethane polymer) may be mixed with other polymers and/or additives to form a polymer composition. Other polymers include polyurethane and polysilicones, which may or may not contain silver or other antimicrobial additives. Suitable additives include fillers, dyes, processing aids and other antimicrobial agents.

The polymer (e.g. polyurethane polymer) or polymer composition may be formed as a coating onto a device, such as a medical device. Alternatively, the polymer or polymer composition may be moulded or cast to form at least part of a device, such as a medical device. Suitable medical devices include implantable devices, such as catheters (e.g tracheal or urethral catheters), pressure sensors and devices used in arterial or venous lines. Other examples of medical devices include dental implants, such as dentures and dental retainers. The polymer or polymer composition may also be formed into a textile, such as a woven or non-woven textile.

EXAMPLES

Materials and methods

Materials

Silver lactate (AgL, Aldrich, UK), Silver sulfadiazine, (AgSD, aldrich UK) and Polytetramethylene oxide (PTMO, M _n ~ 2000, Sigma-Aldrich, UK) were dehydrated at 80°C in vacuum for 24 hours before use. 1,4-butane diol (BD, Sigma-Aldrich, UK), 4,4 '-methylene diphenyl diisocyanate (MDI, Sigma-Aldrich, UK) and dibutyltin dilaurate catalyst (DBTBL, Sigma-Aldrich, UK) were used as received. Tetrahydrofuran (THF, Sigma-Aldrich, UK), Dimethylformamide(DMF, Sigma-Aldrich ,UK) and Dimethylsulfoxide (DMSO, Sigma-Aldrich ,UK) were dried over molecular sieve (4A) before use. Phosphate Buffer Saline (PBS) prepared as follow: disodium hydrogen phosphate (Na ₂ HP0 ₄ , BDH, UK), 52.8 mM,7.50 g/L; sodium dihydrogen phosphate (NaH ₂ P0 ₄ , BDH, UK), 15.6 mM, 1.87 g/L and sodium chloride (NaCl, Sigma, UK), 5.1 mM, 2.98 g/L. All the buffer salts were dissolved in 1 litre of distilled water with pH adjusted to 7.4 by drop-wise addition of concentrated sodium hydroxide solution (5 M NaOH, BDH, UK).

Methods

Polymer synthesis

The silver containing polyurethanes were synthesised via two-steps; solution polymerisation of PTMO, MDI and then chain extension with BD followed by end capping with silver lactate or silver sulfadiazine. The synthesis was carried out in a three-neck flask equipped with a stirrer, a nitrogen inlet and condenser guarded by a calcium chloride drying tube. The reaction was carried out at a molar ratio of (PTMO: MDI: BD: AgL(or AgSD)) of 1 :2:0.9:0.2. AgL (or AgSD) was added to the reactor in final step of the reaction. The temperature of the reaction in the first step was kept at 50°C whilst soft segment was dissolved in DMSO: DMF (1 : 1) mixed solvent. The MDI was also dissolved in the mixed solvent, but added drop-wise to the reactor. Temperature was gradually increased to 80°C and maintained for 1 hour to form the prepolymer. The prepolymer was chain extended with BD followed by adding and silver lactate or silver sulfadiazine. The reaction was carried out for four hours at 120°C. Reaction mixture was cooled down to room temperature and the copolymer was precipitated into propanol/water (1 : 1) solution, then washed with methanol and water several times, filtered and dried in a vacuum oven at 80°C for 24 hour. Polyurethanes with silver lactate and silver sulfadiazine were labelled as PEU-AgL and PEU-AgSD respectively.

Characterization Spectroscopy 'H-NMR analyses of the polymers were conducted using a Burker AV 400 MHz 'H-NMR using CDC1 ₃ as a solvent (Burker Analytik GmbH, Germany).

Raman spectra of the samples were recorded using a Nicolet Almega XR dispersive Micro Raman spectrophotometer (Thermo Fisher Scientific, Madison Wisconsin, USA), equipped with a 785nm laser. All the spectra were collected in the range 3430-100 cm ^"1 using a x lO objectives and over an average of 128 scans, 1 second exposure time.

FTIR spectra of the synthesised polymers were obtained using a Nicolet 8700 FTIR spectrometer (Thermo Electron Corporation, UK) where the polymer sample films were cast on the KBr crystal to obtain spectra. Spectra were recorded in the mid infrared region (4000-400 cm ⁴ ) at 4 cm ^" resolution and averaging 64 numbers of scans.

Mechanical properties

A tensile strength test was performed using an Instron Universal Testing machine (model 5584,Instron Co., UK) equipped with a 10 N load cell at room temperature. Dog bone shape specimens (minimum of 4 samples) were cut from cast films using an ASTM D638 standard punch. The thickness of the films was between 0.1-0.3 mm. The specimens were stretched until break at a crosshead rate of 20mm/min. Young's modulus; Ultimate tensile strength and percent elongation at break were obtained from the stress-strain curves.

Contact Angle

The contact angle of deionised water at the polymer surface was measured by the sessile drop method with a KSV CAM200 contact angle setup (KSV Instruments Ltd, Finland). Contact angle measurement was conducted at room temperature using 5 μΐ liquid drops using a micro-litre syringe. The contact angles of both side of the drop were measured. Separate image frames were collected every 2 seconds for 20 seconds and mean contact angle was calculated using CAM software Molecular weight

Molecular weights of the polymers were obtained with a Zetasizer, nanoseries analyzer (ZS, Malvern Instruments Ltd. Worcestershire, UK) at 25°C using light scattering method according to the refractive index of the polyurethane [20] . Polymer solutions in THF with different concentration were prepared in range of (0.02-0.0025 g/ml), and filtered using 0.2μιη PTFE filter.

Bacterial test

S. aureus was selected as test micro organisms to determine the bactericidal activity of polymers. Bacterial strains for the control were stored at -70°C on beads in glycerol broth. From the beads, the strains were sub-cultured in LB medium every week. From this pure culture, touching at least four morphologically similar colonies, the culture was transferred into Iso-sensitest broth supplemented with 5 % (v/v) defibrinated horse blood. Bacteria were grown aerobically at 37°C for 18 hr. The visible turbidity equal to 0.5 McFarland Standards (BioMerieux, Basingstoke, UK) was achieved by adding sterile distilled water. To aid comparison, the test and standard were compared against a white background with a contrasting black line. The culture was finalized by dilution in the ratio of 1 : 10 using sterile distilled water for S. aureus before inoculation.

For the zone inhibition study, 25 ml of sterile Iso-Sensitest agar according to the manufacturer's instructions was poured in to 90 mm diameter disposable sterilized Petri dishes supplemented with 5 % (v/v) defibrinated horse blood and allowed to solidify. The plates were stored at 4-8°C in sealed plastic bags. 10 μΐ of the bacterial water was streaked over each plate and spread uniformly using a sterile needle of approximately 6 cm length. Pieces of the silver loaded and unmodified PU were gently placed over the solidified agar gel in different Petri dishes for both the bacterial strains. Incubation times were 18-20 hr at 37°C.

Polymer Film Stability

To examine the stability of the Ag-PEU films, polymer films were cut in to 50 mmx50 mmx0.3- 0.4 mm and immersed in PBS for 7 days at 37°C. The films were taken out from the solution, rinsed with distilled water, dried in a vacuum oven at 80°C and 0.1 bar, and then weighed again (Wdry/mai)

W„ dry W„ dry , final

Weight loss (%) x lOO

W„ dry

The mean value of five measurments from different samples is reported.

Film preparation Polyurethane film samples about 0.5-2 mm thick were cast on Petri dishes using 5%-10% (w/v) polymer solution in THF. The Petri dishes were covered loosely, and films dried at room temperature for 72 hours under fume cupboard. The dried films were then removed from the Petri dishes and further dried under vacuum at 0.1 torr and 80°C for 24 hours before testing.

Results and discussion Synthesis of polymers The proposed chemical structures of the modified polymers are shown Fig.l and the results of synthesis (i.e. yield of polymerisation and molecular weight) are given in Table 1. AgL and AgSD were insoluble in most of the organic solvents; hence the synthesis was carried out in highly polar solvents such as DMF and DMSO. The molar ratio of polymers was calculated in a way to avoid cross-linking (1 : 1 for (diols and AgSD or AgL) : diisocyanates). During the polymerisation mixture of DMF and DMSO was added in large excess to dissolve the linear polymer. In addition filtration was carried out in order to separate out any cross linked polymer formed, hence linear polymers were obtained.

Table 1. Molecular weight, yield of reaction and silver content of synthesized polymers

The molecular weight of the modified polyurethanes and unmodified material are approximately similar. However, the AgL containing polymer showed lower and yield of reaction compared with AgSD end capped polymer, which could have been due to a limited solubility of the AgL in the organic solvents.

(b)

Fig. 1. Chemical structure of (a) PEU-AgL (b) PEU-AgSD

Polymer characterization Spectroscopy

The H'NMR spectra of the modified polymers are shown in Fig 2. The urethane N-H proton peak is located at δ=8.64 ppm, confirming the polymerization. MDl reacted with diols and AgL (or AgSD) and the urethane linkage is formed. Additional peaks at δ= 7.48, 7.15 ppm can be attributed to the aromatic protons of MDl and the peak at 5=3.81 ppm is assigned for methylene from MDL Peaks at δ=4.88, 1.42 ppm in PEU-AgL, are showing proton in (CH and CH ₂ ) silver lactate[21] and the peak at δ= 9.2 ppm in PEU-AgSD assigned for methylene group in silver sulfadiazine.

The Raman spectra of polyurethane samples (Fig. 3) show peaks at 1303,1250 and 1185 cm ^"1 corresponding to urethane amide [22]. Furthermore the bands at 225, 429 cm ^"1 are assigned to the symmetric bending mode of Ag ⁺ in both AgL and AgSD containing samples. Raman spectrum could confirm that not only the polymerization and end capping reactions were successful but silver is stable in coordinate with lactate and sulfadiazine groups. The FTIR spectra of the polymers (Fig 4.) show no cyanate group (N=C=0) peak at 2350-2275 cm ¹ indicating that the reaction went to completion. Urethane formation confirmed by the N-H stretching peak at 3320 cm ^"1 and urethane carbonyl at 1703 and 1730 cm ^"1 . The symmetrical and asymmetrical stretch of the methyl and methylene group is observed at 2935 and 2856 cm ^"1 , respectively. Hydrogen bonded C=0 group showed signal at 1703 cm ^"1 and stretch and bending of N-H with C-N is appeared at 1530 cm ^"1 . The peak at 1110 cm ^"1 represents the C-O-C bond in polyurethane. In general, formation of polyurethane and end capping of linear polymer with AgL and AgSD is verified with NMR, Raman and FTIR spectroscopy.

The surface morphologies of the silver modified films (Fig.5) did not show a significant change on surface texture of polyurethane. Samples were also subjected to a qualitative X-ray energy dispersive spectrum. This was to verify and confirm the presence of silver in polyurethane films. EDX measurements were performed on both PEU-Ag films indicating the existence of the silver on surface of both films (data not shown).

Mechanical testing

The mechanical testing results of synthesised polymers are presented in table 2. Elastic modulus, ultimate tensile strength and elongation at break were determined from stress-strain plots for each sample.

Table 2. Mechanical test (tensile test) results of polyurethane and samples end capped with silver sulfodiazine and silver lactate (n=4).

Tensile test results showed, end capping polyurethane with silver lactate and sulfadiazine did not affect its mechanical properties. The slight decrease in elongation at break and tensile strength of silver containing samples could be because of the lower molecular weight of the modified polymers. The elastomeric behaviour of these polymers could be an advantage for coating applications particularly for devices with irregular shapes as conformal coatings are more likely.

Water contact angle Hydrophilicity is an important factor for bacterial adhesion and biocompatibility of materials [23, 24]. As shown in Fig.6 the silver containing polymers had lower water contact angle compared with control polyether-urethane. It is clear that hydrophilicity of polyurethane increased with incorpration of both silver lactate and silver sulfadiazine. This hydrophilicity increase of polyurethane with silver complex incorporation could be due to either presentation of silver ion/metal or the chelating groups ( lactate and sulfadiazine) which may lead to increased water absorption by the polymer. The percentage of weight loss of polymer films was calculated after immersion in phosphate buffer saline for 4 weeks (Fig. 7). Results showed that the weight loss of Ag containing polyurethane at 37°C is almost the same as control polyether-urethane. The modified polymers did not show additional sign of leachables and all polymers were stable in buffer solution; also there is no sign of significant weight loss.

The silver modified films' antibacterial activities were assessed by placing in Petri dishes and measuring zone of inhibition by utilising agar diffusion tests. Distinct differences in the membranes tested using S.aureous were noted. The PEU-AgSD membrane exhibited an inhibition zone which extended approximately 3mm from the edge of the modified polymer membrane. Furthermore, the bacterial growth beneath the PEU-AgL membrane was found to be poor when the membranes were lifted off the agar plate. This could imply that there is a direct inhibitory effect of the membrane on bacterial growth, even though no inhibition zone was observed. Contrastingly, unmodified PEU supported the growth of bacteria to some extent. Difference of inhibition may have been due to differences of silver concentration on surface or silver ions retained in the membrane. It is also important to adjust the silver ion concentration in membranes to achieve effective antibacterial properties.

Conclusion

In this study the possibility of synthesising a bacterial resistant elastomeric polymer by reaction of silver lactate and silver sulfadiazine with MDI (end group in functionalized polyurethane) in solution has been reported. The spectroscopy data (FTIR, Raman and NMR) confirmed that the new polymer has been synthesised successfully. In addition, the mechanical properties of modified polymers did not show a significant change and polymers were stable in vitro. However the silver content of the membranes were low, acceptable antibacterial activity was observed. Further studies are essential to develop knowledge about the novel polymer introduced in this research.

The methodology which employed in this study shows that antibacterial biomaterials from silver loaded polymers to prevent bacterial colonization could be achieved without change in mechanical properties of conventional materials.