PROCESS

The present invention relates to a process for the production of a polymer-metal complex.

Combinations of certain metals with polymers have shown considerable potential for use as bioactive materials and/or for their catalytic applications. US 5817325 and US 5849311, for example, provide examples of antimicrobial articles, devices and formulations prepared by applying an organic coating to a substrate followed by immersing the coated substrate in a solution of silver or potassium salt.

However, in many of the polymer-metal combinations disclosed in the prior art, the metal is present in its ionic state as part of either an inorganic or an organic salt. The use of polymer-metal combinations in this form presents a number of problems, for example there is limited control over the release of the metal ions, which results in a rapid decrease in its effectiveness and, in the case of biomedical applications, there is the possibility of excess metal ions entering the body. Further, in the case of silver, staining of the skin on contact with silver ions and a change in colour of the material due to gradual photoreduction of the silver ions are additional problems.

These problems have been overcome by reducing the metal ions to form polymer- metal complexes. In this respect several processes have been disclosed in the prior art, see, for example, JP 04 002877 and JP 03 045709, which describe the production of metal colloids by chemically reducing water-soluble salts of copper, lead, silver or tin in an aqueous solution containing a water-soluble polymer, or a mixture of water-soluble polymers thereof.

WO 00/49219 discloses a process for treating a substrate to impart biocidal properties. The process comprises the steps of depositing or impregnating the substrate with solubilised chitosan, immersing the substrate in a solution of a silver salt and reducing the silver salt to atomic/metallic silver. Likewise, WO 02/15698 and EP-A- 1312262 also describe the preparation of articles or powders, respectively, having contact biocidal properties.

Although WO 00/49219, WO 02/15698 and EP-A-1312262 describe processes for the production of certain polymer-metal compositions (e.g., chitosan-silver compositions), it is clear from the physical, chemical and biological properties of these compositions that they contain considerable proportions of unreduced silver ions, despite using both photochemical and complex chemical reducing methods and, in some cases elevated temperatures, for the reduction step.

There remains a need for improved processes for producing polymer-metal complexes and to improved polymer-metal complex products.

In a first aspect of the present invention, there is provided a process for producing a polymer-metal complex comprising the steps of:

(i) contacting a solution comprising one or more metal salts with a polymer which is capable of interacting with the metal salt to form a polymer-metal ion complex and wherein the polymer and the polymer-metal ion complex are in the solid phase, said solid phase comprising the polymer in an amount of at least 25% by weight and/or being in the form of a powder or flake;

(ii) optionally dissolving the polymer-metal ion complex; and (iii) chemically reducing metal ions in the polymer-metal ion complex to form the polymer-metal complex.

In a second aspect, the invention also provides the product obtainable by the process of the first aspect of the present invention.

In a third aspect, there is provided a polymer-metal complex in solution, which is translucent and free of suspended particulate metal-containing species. Thus, preferably, the solution contains no colloidal and/or suspended particles.

In a fourth aspect, there is provided a solid premix comprising a polymer-metal ion complex and a solid reducing agent.

In a fifth aspect, the invention provides a process for the production of a polymer- metal complex comprising the steps of:

(a) contacting an aqueous solution comprising one or more metal salts with a solution comprising alginic acid or a salt thereof, polyvinylamine or a mixture thereof;

(b) treating the solution in (a) with a gelling agent to form a gel; and

(c) chemically reducing metal ions in the gel, wherein (b) and (c) may be carried out simultaneously or sequentially in any order.

In a sixth aspect, the invention provides the use of a reductone for reducing a polymer-metal ion complex to a polymer-metal complex.

In a seventh aspect, there is provided a disinfectant composition comprising the polymer-metal complex produced according to the process of the first or fifth aspects of the invention.

In the eighth aspect, the invention provides the use of the polymer-metal complex produced according to the process of the first or fifth aspects of the invention as a non-migrating anti-microbial agent.

The polymers useful in the process of the present invention, and which may be employed in step (i), preferably comprise functional groups selected from amine, carboxylic acid, carboxylate or hydroxyl, and mixtures thereof; and may be natural or synthetic polymers. For example, the polymer may be selected from the group consisting of chitin, chitosan and polyvinylamine, poly(ethylene imine), ρoly(lysine), 'cationic' polyacrylamide copolymers, cellulose that has been derivatised to incorporate amine groups, alginate, hyaluronic acid, carboxymethyl cellulose, and polymers and copolymers containing acrylic acid and similar monomers. Most preferably, however, the polymer used in. step (i) comprises amine functional groups and is selected from the group consisting of chitin, chitosan and polyvinylamine. Particularly preferred polymers are chitin and chitosan.

Chitin is a natural polymer found in the exoskeleton of crustaceans, the internal pen of squid, and the cell wall of a number of fungi. It is most commonly used after conversion to chitosan by a deacetylation treatment. Although chitosan may be obtained directly from certain fungi, almost all commercial chitosan is currently obtained from crustacean-derived chitin. However, chitin and chitosan from fungal sources are gradually becoming available in commercial quantities. The terms 'chitin' and 'chitosan' do not refer to discrete and distinct polymers but to a range of structurally closely related polymers differing in the ratio of D- glucosamine andiV-acetyl-D-glucosamine residues. Chitins normally contain from 5-10% D-glucosamine residues and all chitosans, unless specially prepared by techniques involving multiple deacetylation processes, contain a proportion of N- acetyl-D-glucosamine residues.

The polymer used in step (i) of the process is a solid and is preferably in the form of a powder or flake. The solid phase typically comprises the polymer in an amount of at least 25%, preferably at least 30%, such as at least 40% or at least 50% by weight, more preferably at least 60% by weight, such as at least 70% or at least 80% or at least 90% by weight. The polymer is typically not in the form of a coating applied to a substrate.

The process steps (i), (ii) and (iii) may independently be carried out at any temperature, such as a temperature between room temperature and an elevated temperature (i.e., above room temperature) such as at a temperature of 25 to 95 ⁰ C or 25 to 100 ⁰ C, more preferably at a temperature of 40 to 100 ⁰ C, such as 40 to 95 ⁰ C, 45 to 9O ⁰ C, 60 to 95 ⁰ C, 60 to 9O ⁰ C or 70 to 90 ⁰ C, most preferably at a temperature of 80 to 90 ⁰ C. Preferably, step (i) is carried out at elevated temperatures, as described above. Most preferably, step (i) is carried out at a temperature of 75 to 85 ⁰ C and the subsequent steps (ii) and (iii) are carried out at room temperature. The term 'room temperature' as used herein has, its ordinary meaning and typically ranges from 21 to 25 ⁰ C. It has surprisingly been found that carrying out step (i) at elevated temperatures gives an improved product and/or increases the stability of a subsequently formed solution of polymer-metal complex.

Without wishing to be bound by theory, it is believed that during step (i), metal ions in the solution are adsorbed, complexed or otherwise associated onto the polymer to form a polymer-metal ion complex. Typically, adsorption takes place at specific sites of the polymer substrate, for example, on the amine groups which may act as ligands and bind directly with the metal ions. Another suitable site for adsorption of the metal ions onto the polymers includes carboxylate groups, where the counter ion, which is usually a sodium, potassium or ammonium ion, can be exchanged for the metal ions in solution. In this case, the binding may be through ionic/electrostatic forces or co-ordination bonds or a combination of these two mechanisms.

Step (i) is preferably performed to the stage of complete absorption, i.e., either when all of the metal ions in solution have been adsorbed onto the polymer or when the polymer is saturated and no more metal ions can be adsorbed. However, it will be appreciated that the rate of adsorption, and consequentially the time taken for complete adsorption, may vary depending on the temperature and other process variables used, such as the concentration of the metal salt solution. Although the solution of one or more metal salts in step (i) can be of any concentration, higher concentrations are preferred.

If not all of the metal ions in solution have been adsorbed onto the polymer during step (i), the polymer-metal ion complex produced may be filtered off and subsequently washed to remove any excess metal salts. This may be carried out in the same solvent for the solution used in step (i), without the metal salts dissolved therein.

The amount, by weight of polymer, of metal ions that is preferably used in step (i) of the process ranges from 0.1 to 20 %, such as 1 to 20%, more preferably from 2 to 15 %, such as 2.5 to 10 %, most preferably from 3 to 8 %.

The one or more metal salts are preferably selected from the salts of silver, copper, gold, palladium, platinum and tin, and mixtures thereof. Silver salts are preferred.

Preferably, the one or more metal salts are present in an aqueous solution, in which case the metal salts must be water-soluble to some extent. Most preferably, the metal salt is silver nitrate. The term aqueous solution as used herein describes a solution in a solvent comprising at least 5%, such as at least 10%, preferably at least 25%, such as at least 50% or at least 75%, most preferably at least 95% such as at least 99% or even 100%, by weight of water.

In an alternative embodiment, the one or more metal salts may be present in a nonaqueous solution, such as a solution of the one or more metal salts in an anhydrous polar organic solvent e.g., a Cχ-Cβ alcohol such as ethanol, methanol or zsopropyl alcohol.

The polymer-metal ion complex formed in step (i) may be dissolved in step (ii), preferably at a pH below 6.5, before the metal ions are subjected to chemical reduction in step (iii). Suitable acids for carrying out step (ii) include but are not limited to organic acids, such as, for example, acetic acid, lactic acid, glutamic acid and glycolic acid.

Although, in one embodiment of the invention, step (i) is limited to contacting a solution comprising one or metals salts to a polymer wherein the polymer and the polymer-metal ion complex produced remain in the solid phase, it will be appreciated that the formation of polymer-metal ion complexes can also be achieved if the polymer is present as part of a solution in step (i). In this case, the resulting polymer-metal ion complex remains in solution and step (ii) of the process is made redundant.

Therefore, the present invention also contemplates a process for the production of a polymer-metal complex comprising the steps of:

(I) contacting a solution comprising one or more metal salts with a polymer which is capable of interacting with the metal salt to form a polymer-metal ion complex and wherein the polymer and the polymer-metal ion complex are in solution; and

(II) chemically reducing metal ions in the polymer-metal ion complex to form the pofymer-metal complex.

Particularly preferred polymers for use in step (I) include water-soluble polymers, such as, for example, polyvinylamine, alginic acid or a salt thereof, carboxymethyl cellulose or a salt thereof, hyaluronic acid or a salt thereof, poly(ethylene imine) or mixtures thereof. However, polymer substrates which require a pH of below

6.5 in order to dissolve in the solution comprising one or metal salts in step (I) are also encompassed in this embodiment of the invention. A pH of below 6.5 may be achieved by the addition of, for example, an organic acid such as acetic acid, lactic acid, glutamic acid or glycolic acid.

Chemical reduction of the polymer-metal ion complex in the process of the invention may be carried out using any suitable reducing agent. Preferably, however, chemical reduction is carried out using a reductone or a salt thereof (e.g., sodium, potassium or calcium salts), such as ascorbic acid or a salt thereof, which are more preferably non-toxic and readily available.

As used herein, the term reductone includes compounds containing an enediol structure stabilised by conjugation and hydrogen bonding with an adjacent group, RC(OH)=C(OH)C(=O)R'. Preferably R and R' are the same or different and are selected from: hydroxyl; C ₁ to C ₂₀ alkyl, optionally substituted with C ₁ to C ₃ alkyl, hydroxyl or C ₁₂ to C ₂₄ acyloxy; and R and R' may be linked to form a ring structure. Reductones are commonly derived from saccharides by oxidation at the carbon atom alpha to the carbonyl functionality. Ascorbic acid and its salts are the preferred reductones, used alone or in combination. Suitable salts of ascorbic acid include food grade salts (such as, for example, sodium, potassium or calcium salts). Sodium salts are preferred.

It will be appreciated that chemical reduction may be performed on polymer-metal ion complexes, which are either in the solid phase or in solution. For example, if the polymer-metal ion complex is in the solid phase (e.g., if step (ii) is omitted from the process), chemical reduction of metal ions may be carried out on the

solid polymer-metal ion complex or the polymer-metal ion complex may be dissolved to form a solution prior to the step of chemical reduction.

Since ascorbic acid aids the dissolution of certain polymer substrates, such as chitosan, the use of ascorbic acid as the reducing agent may form part or all of the acid required to perform steps (ii) and (iii) of the process, simultaneously.

The molar ratio of reducing agent to metal ions in the polymer-metal ion complex in the chemical reduction step is preferably from 2:1 to 1 :2.

During the chemical reduction step (i.e., step (iii) or step (H)), metal ions in the polymer-metal ion complex are reduced. Preferably, the metal ions are reduced to metallic atoms to form a polymer-metal complex. Surprisingly, the use of a reductone in step (iii) or step (II) provides an advantage in that substantially complete reduction of the metal ions in the polymer-metal ion complex is achieved, as evidenced by the absence of a zone of inhibition in the biocidal activity test described in Example 22. Example 22 demonstrates that unlike the products obtained by the processes described in WO 00/49219, WO 02/15698 and EP -A-1312262, which behave as migrating anti-microbial agents, those obtained by the processes descried herein are true non-migrating anti-microbial agents.

Preferably, the product of the chemical reduction step comprises more than 0.1% by weight of metal in the polymer-metal complex, more preferably more than 0.5%, such as more than 1.5%, most preferably more than 3.5%, such as more than 6% or more than 10% or more than 15%. Typically, the product contains less than 50%, such as less than 25%, by weight of metal in the polymer-metal complex.

If step (ii) is omitted, the polymer-metal complex formed in step (iii) may be in solid form. This solid can be filtered off, washed and dried. Washing may be carried out using an aqueous solution. Alternatively or subsequently, the solid polymer-metal complex may be dissolved at a pH below 6.5, ^" using the same acids mentioned above in relation to step (ii).

Solutions of polymer-metal complex produced by the present invention may be treated to precipitate out the polymer-metal complex (e.g., by neutralising the solution), which can then be washed using an aqueous solution and subsequently dried. Alternatively, the solution of polymer-metal complex is spray dried to form solid polymer-metal complex in the form of a free flowing powder.

The polymer-metal complexes produced by the present invention may be used in sprays or aerosols, or used as a coating material, or formed into fibres, films, foams, freeze-dried mats, gels, hydrogels or sponges. In addition, the polymer- metal ion complexes may also be used as a coating material, or formed into fibres, firms, solid foams, freeze-dried mats, gels, hydrogels or sponges prior to the chemical reduction step.

It has surprisingly been found that solutions of the polymer-metal complex produced by the process according to the invention are typically homogeneous solutions. Whereas polymer-metal ion complexes (e.g., polymer-silver ion complexes) show negligible absorption across the visible spectrum, chitosan-silver complexes formed by the process as described above, in relation to the first aspect of the invention, show a strong absorption band in the region 400-420 nm. The extinction coefficient for polymer-metal complexes (e.g., polymer-silver complexes), measured in a 1 cm cell, based on the molar concentration of silver present, is preferably greater than 10,000 L mol ^"1 cm ^"1 .

For example, if the chemical reduction step (i.e., step (iii) or step (II)) is performed on polymer-metal ion complexes which are in solution, the solution of polymer-metal complex produced is typically clear and has good stability, with a shelf life generally in excess of three months. This is in contrast with the solutions obtained following the processes disclosed in

EP-A-1312262 and WO 02/15698 (involving photochemical reduction, only), which are typically opaque and colloidal in nature, either on initial formation or rapidly become so on standing. Furthermore, in comparison with the solutions produced by the invention, these solutions have a much reduced shelf life in which a precipitate typically settles out after one or two weeks.

The difference between the solutions produced by the invention and those mentioned above may be quantified by consideration of the extinction coefficients of the polymer-metal complexes. For example, a solution of chitosan-silver ion complexes is ordinarily more or less colourless (apart from any slight colour due to impurities in the chitosan) but on reduction to form chitosan-silver complexes, the solution becomes brown in colour. Generally, this colour gives rise to an absorption band at around 400 urn. The intensity of this absorption band, together with the concentration of the silver, can be used to calculate the extinction coefficient for the chitosan-silver complex. As mentioned above, the values obtained for solutions of polymer-metal complexes produced by the process of the invention (measured in a 1 cm cell, based on the molar concentration of silver present) are typically around (preferably greater) than 10,000 L mol ^"1 cm ^"1 . In contrast, the values obtained for solutions of polymer-metal compositions produced following the methods disclosed in EP-A-1312262 or WO 02/15698 are typically around 2,500 L mol ^'1 cm ^"1 .

Without wishing to be bound by theory, the difference in the extinction coefficient values is thought to be due to a combination of at least two factors. The first factor is believed to be due to the difficulty in obtaining complete reduction of the metal ions in the polymer-metal ion composition by photochemical means. In particular, the extinction coefficient value of about 2,500 L mol ^"1 cm ^"1 was obtained after 72 hours of photochemical reduction, which suggests that the product contains a mixture of silver ions and silver in the form of metallic atoms. Thus, it is believed that a lower proportion, by weight, of the metal ions in the polymer-metal ion compositions are reduced to metallic atoms during the photochemical reduction step of EP-A-1312262 and WO 02/15698 compared with the process of the invention.

Secondly, the opaque nature of the solutions produced in accordance with EP-A- 1312262 or WO 02/15698 suggests that the silver is aggregated rather than being present as discrete atoms. This aggregation is also thought to reduce the value of ^■ the extinction coefficient. The fact that the absorption band at around 400 nm is typically much broader for these solutions compared with the solutions produced by the invention supports the theory of aggregation.

Following the addition of a reductone (e.g., ascorbic acid) to the solutions produced by the processes of EP-A-1312262 and WO 02/15698, the extinction coefficient generally increases to a value of around 6,000 L mol ^"1 cm ^" . This provides further support that the product produced by photochemical reduction is both incompletely reduced and that the silver atoms/compounds are aggregated.

If the chemical reduction step (i.e., step (iii) or step (II)) is performed on polymer- metal ion complexes (e.g., polymer-silver ion complexes) in the solid phase, the polymer-metal complex produced is typically light to very dark brown in colour depending on the concentration of silver present. In contrast, the polymer-metal composition obtained following the process disclosed in EP-A- 1312262 varies in colour from purple-silver, to brown, to yellow depending on whether the halide ion is chloride, bromide or iodide, respectively. Without wishing to be bound by theory, this suggests that the product obtained by subjecting the polymer-metal ion complexes to photochemical reduction (as disclosed in EP-A- 1312262, WO 02/15698 and WO 00/49219, for example) contain particles of aggregated silver compounds that are held physically within the chitosan but not attached to it, rather than being present generally as single silver atoms complexed or otherwise associated at specific sites of the polymer substrate (e.g., at the amine groups of chitosan).

In order to improve the clarity and/or the shelf-life (stability) of the solutions produced by the process of the present invention a polymer-compatible surfactant may be added before or after steps (ii) or (iii), and before or after step (II). For example, cationic and non-ionic surfactants may be used in conjunction with chitosan and other polymers that are positively charged in solution, while anionic and non-ionic surfactants may be used in conjunction with alginate and other anionic polymers.

Thus, in a preferred embodiment of the process, prior to the step of chemically reducing metal ions in the polymer-metal complex, the polymer-metal ion complex may be contacted with a surfactant, preferably, a quaternary ammonium compound, such as a quaternary ammonium compound having an alkyl chain of from 6 to 24 carbon atoms, for example cetyl trimetliylammonium bromide. The

addition of such a compound surprisingly produces a solution which, after chemical reduction and, if required,- dissolution of the polymer-metal complex, is less coloured than solutions otherwise produced.

Depending on the intended use, it may be desirable to insolubilise the resulting polymer-metal complex by cross-linking or other means known in the art. In order to maintain the solubility characteristics of the polymer in the resulting polymer-metal complex, preferably, no cross-linking or equivalent step is performed.

The present invention also provides a polymer-metal complex in solution, which is translucent (i.e., typically non-opalescent) and free of suspended particulate metal- containing species. This solution has unexpectedly been found to be produced by the invention. Preferably, the solution is a clear (i.e., transparent) solution.

The present invention also provides a solid premix. By the term solid premix, it is meant a mixture or blend of two or more components in the solid phase. Preferably the premix is in the form of a powder.

The polymer-metal ion complex contained in the solid premix may be produced according to step (i) of the process, as described above. However, it is also contemplated that the polymer-metal ion complex may be produced according to step (I). In this latter case, the resulting solution of polymer-metal ion complex is treated to obtain the polymer-metal ion complex in the solid phase. This may be achieved using any suitable technique known in the art but is preferably performed by spray drying or a method used to induce precipitation of the solid out of solution.

The solid premix comprises a solid reducing agent. Preferably, the reducing agent is acidic, such as, for example, solid ascorbic acid or a salt thereof or mixtures thereof. Additionally, the premix may further comprise a solid acid, such as glycolic acid, in addition to the polymer-metal ion complex and reducing agent. Since ascorbic acid acts as a reducing agent and aids the dissolution of certain polymer substrates (e.g., cbitosan), the use of a solid acid in addition to the

polymer-metal ion complex may be omitted if ascorbic acid is used as the reducing agent. It will be appreciated that if ascorbic acid is used as both a reducing agent and solubilising agent, the ascorbic acid is preferably present in a higher concentration than if it were acting solely as a reducing agent.

One advantage of forming a premix that is solid is the ease with which it can be transported and handled. By subsequently dissolving the solid premix, the metal ions in the polymer-metal complex can be reduced, when required. Another advantage is the greater storage life of the product in premix form compared with the storage life of the product in solution form. Also, providing the product in premix form provides greater versatility as the user may readily produce solutions of varying concentrations from the same batch of solid premix.

According to the process of the fifth aspect of the present invention, steps (b) and (c) may be carried out simultaneously. Therefore, the process may comprise, after step (a), contacting the product formed in (a) with an agent, such as calcium ascorbate, that causes both gelling and chemical reduction to take place.

Alternatively, steps (b) and (c) may be carried out sequentially and in either order, i.e., step (b) may be carried out before step (c) or, alternatively, step (c) may be carried out before step (b).

Suitable salts of alginic acid include sodium, potassium, magnesium and mixtures thereof. Preferably, sodium alginate is used in step (a).

The gelling agent used in step (b) preferably comprises calcium ions. Suitable gelling agents used in step (b) preferably include any water-soluble salt of calcium, such as calcium chloride, calcium nitrate, calcium ascorbate and mixtures thereof.

The aqueous solution comprising one or more metal salts in step (a) may be selected from the salts of any metal but are preferably selected from the salts of silver, copper, gold, palladium, platinum and tin, and mixtures thereof.

During step (a), metal ions are adsorbed, complexed or otherwise associated onto the alginic acid or salt thereof and/or the polyvinylamine to form an alginate/polyvinylamine-metal ion complex as described above in relation to step

G ⁾ .

Chemical reduction of metal ions in the gel may be carried using any suitable reducing agent; however, the use of a reductone is preferred, in particular ascorbic acid or a salt thereof. Suitable salts of ascorbic acid include sodium, potassium and calcium salts. Sodium salts are preferred.

The present invention also provides the use of a reductone for reducing a polymer- metal ion complex to a polymer-metal complex. Preferably, the metal ions are reduced to metal atoms. In the preferred embodiment, the reductone is preferably ascorbic acid or a salt thereof. It will be appreciated that the use of a reductone for reducing polymer-metal ions can be carried out as described above in relation to step (iii), step (II) or step (c) of the processes contemplated by the present invention.

The products prepared by the processes of the present invention may be used as bioactive materials, particularly in the biomedical and/or healthcare fields. Also, due to the extremely reactive nature of the metal in the polymer-metal complexes these products can be used as catalysts in a number of chemical and/or biochemical processes.

In a seventh aspect, there is provided a disinfectant composition comprising the polymer-metal complex produced according to the process of the first or fifth aspects of the invention. The polymer-metal complex typically acts as a non- migrating anti-microbial agent

Preferably, the disinfectant composition is for use as a hand or skin disinfectant, which may ideally be used in the food industry or in hospitals. The disinfectant composition may be in solid or liquid form and may take the form of a gel, hydrogel, cream, solution, emulsion, foam, aerosol spray, bar or any other suitable

formulation known in the art. Preferably, the disinfectant composition is in the form of a gel or cream.

Examples

Example 1

1O g of finely ground chitosan particles was stirred in 70 ml deionised water. 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise into the chitosan suspension. The chitosan suspension was stirred until the supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was then diluted to 970 ml with deionised water and 9 ml lactic acid (80%) was added to solύbilise the chitosan/ Ag ⁺ complex. Once a homogeneous solution was obtained a solution of 0.9 g ascorbic acid in 20 ml deionised water was added. A strong yellow-brown colour developed rapidly and full reduction was achieved within 1 hour.

Example 2

1O g of finely ground chitosan particles was stirred in 70 ml deionised water while 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to it. The suspension was stirred until the supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was diluted by the addition of 30 ml deionised water and stirred while 1 g sodium ascorbate in 25 ml deionised water was added.

The particles of chitosan rapidly acquired a dark brown colour indicating that the silver ions had been reduced and after 1 hour the suspension was filtered. The solid chitosan/Ag ⁰ was washed with deionised water and dried at 60 ⁰ C. A solution of 1 g of the dry powder in 100 ml of 0.9% lactic acid (80%) gave an extinction coefficient of 10,600 L mol ^"1 cm ^"1 for the chitosan/Ag ⁰ complex, based on the molar concentration of silver present.

Example 3

1O g of finely ground chitosan particles was stirred in 70 ml deionised water while 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to it. Stirring continued

until the supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was then diluted to 800 ml with deionised water, and 16g ascorbic acid dissolved in 200 ml deionised water added. Reduction and solubilisation occurred concurrently to give a strongly coloured (yellow-brown) solution with an extinction coefficient > 10,000 L moF ¹ cm ^"1 , based on the molar concentration of silver present.

Example 4

1O g of finely ground chitosan particles was stirred in 70 ml deionised water while 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to it. Stirring continued until the supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was filtered and the recovered solids dried at 65 ⁰ C. The dried powder was intimately blended with glycolic acid powder (7 g) and ascorbic acid powder (1 g). 4.5 g of the powder was weighed out into a beaker, 250 ml deionised water added and the mixture rapidly stirred. Reduction and solubilisation occurred concurrently to give a strongly coloured (yellow- brown) solution with an extinction coefficient > 10,000 L mol ^"1 cm ^'1 , based on the molar concentration of silver present.

Example 5

A lO g sample of finely ground chitosan particles was stirred in 70 ml deionised water while 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to it. Stirring continued until the supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was then filtered and the recovered solids dried at 65 ⁰ C. The dried powder was intimately blended with ascorbic acid powder (18 g). 7 g of the powder was weighed out into a beaker, 250 ml deionised water added and the mixture rapidly stirred. Reduction and solubilisation occurred concurrently to give a strongly coloured (yellow-brown) solution with an extinction coefficient > 10,000 L mol ^"1 cm ^" l, based on the molar concentration of silver present.

Example 6

10 g of finely ground chitosan particles was stirred in 70 ml deionised water and

1.2 g AgNO ₃ in 10 ml deionised water added drop wise to it. The chitosan suspension was stirred until the filtered off supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was then diluted to 950 ml with deionised water and 9 ml lactic acid (80%) was added to solubilise the chitosan/Ag ⁺ complex. Once a homogeneous solution was obtained a solution of

1.8 g ascorbic acid in 40 ml deionised water was added. A strong yellow-brown colour developed rapidly and full reduction was achieved within 1 hour.

Example 7

1O g of finely ground chitosan particles was stirred in 70 ml deionised water while 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to it. Stirring continued until the filtered off supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was diluted by the addition of 30 ml deionised water and stirred while heating to 70-75 ⁰ C. On reaching the desired temperature 1 ml hydrazine (98%) was added and stirring continued at this temperature for 1 hour then the suspension was filtered, the solid chitosan/Ag ⁰ washed with deionised water and dried at 60 ⁰ C. A solution of 1 g of the dry powder in 100 ml of 0.9% lactic acid (80%) gave an extinction coefficient of

10,200 L mol ^"1 cm ^"1 for the chitosan/Ag ⁰ complex, based on the molar concentration of silver present.

Example 8

A sample (5 g) of chitosan/Ag ⁰ powder prepared as in Example 2 was suspended with stirring in 100 ml of water. Once the particles of chitosan/Ag ⁰ were thoroughly dispersed, 3 ml lactic acid (80%) was added and the mixture stirred until a viscous solution was formed. This was then transferred to a syringe and the solution extruded into the bottom of a vertical tube containing 1 litre of a 2% NaOH solution. The stream of polymer solution gelled and gradually rose towards

the top of the tube where it was removed and rinsed in several changes of water and then dried to give a brown fibrous product.

Example 9

1O g of finely powdered chitirx having approximately 8% B-glucosamine residues was slurried in 80 ml de-ionised water containing 0.3 g AgNO ₃ until the filtered off supernatant did not give any precipitate on addition to a solution of NaCl. A solution of 0.3 g ascorbic acid in 10 ml deionised water was added and stirring continued for 1 hour, during which time the powder developed a dark brown colour. The solid was filtered off, rinsed well and dried.

Example 10

A solution containing 0.3 g AgNO ₃ and 8 g sodium alginate in 200 ml deionised water was left to stand for 24 hours in the dark. Then the solution was stirred while 10 ml of a 3.5% solution of ascorbic acid was slowly added. The solution rapidly developed a grey colour, indicating that the silver had been reduced.

Example 11

The solution prepared in Example 10 above was, before reduction of the complexed silver ions, transferred to a syringe and the solution extruded into the bottom of a vertical tube containing 1 litre of a dilute solution of a soluble calcium salt. The stream of polymer solution gelled on contact with the calcium ions and gradually rose towards the top of the tube where it was removed. The gel was rinsed with several changes of water, immersed in a dilute solution of ascorbic acid or sodium ascorbate, and then rinsed and dried to give a grey fibrous product that from visual inspection had a uniform colour.

Example 12

The solution prepared in Example 10 above was, before reduction of the complexed silver ions, transferred to a syringe and the solution extruded into the

bottom of a vertical tube containing 1 litre of a dilute solution of a soluble calcium salt and ascorbic acid or sodium ascorbate. The stream of polymer solution gelled and gradually rose towards the top of the tube where it was removed and rinsed with several changes of water and then dried to give a grey fibrous product that from visual inspection had a uniform colour. Thus, the reduction step may be combined with the coagulation/gelation step.

Example 13

A homogeneous solution of the chitosan/Ag ⁺ complex, prepared as described in Example 1, was heated to 70-75 ⁰ C and 1.0 ml hydrazine (98%) in 5 ml deionised water added, followed by a further 1.5 ml lactic acid (80%) to redissolve the precipitated chitosan-silver complex. There was a rapid development of an intense yellow-brown colour, which on cooling back to room temperature showed an extinction coefficient similar to that of the product obtained in Example 1.

Example 14

To a solution of the chitosan/Ag ⁺ complex, as prepared in Example 1, was added 5 g of a non-ionic surfactant (Tween 20) followed by 0.9 g ascorbic acid in 20 ml deionised water. The stirred solution rapidly changed from colourless to a strong reddish-brown colour, indicating that reduction had taken place. The shelf life of this solution was found to be greater than that of a solution prepared similarly but with omission of the surfactant.

Example 15

A solution containing 10 g poly(vinylamine) in 500 ml deionised water was stirred while 0.6 g AgNO ₃ in 10 ml deionised water was added. After standing overnight the solution was stirred while a solution of 0.9 g ascorbic acid in 20 ml deionised water was added. An intense yellow-brown colour rapidly developed and reduction was complete within 1 hour.

Example 16

10 g of finely ground chitosan particles in 100 ml deionised water was stirred while heated to 80-85 ⁰ C. Once this temperature was reached 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to the suspension and stirring continued at

80-85 ⁰ C for 1-2 hours. The mixture was diluted to 980 ml with cold deionised water and 9 ml lactic acid (80%) added to solubilise the chitosan/Ag ⁺ complex.

Once a homogeneous solution was obtained, 0.9 g ascorbic acid in 10 ml deionised water was added and stirring continued for 1 hour, by which time the solution had developed a strong reddish-brown colour. The solution gave an extinction coefficient at 400 run, based on the molar concentration of silver present, of > 10,000 L mol ^'1 cm ^"1 .

Example 17

10 g of finely ground chitosan particles in 100 ml deionised water was stirred while heated to 80-85 ⁰ C. Once this temperature was reached 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to the suspension and stirring continued at 80-85 ⁰ C for 1-2 hours. The suspension was then filtered and the recovered solids dried. The dried powder was intimately blended with 7 g of glycolic acid powder and ascorbic acid powder. 4.5 g of the mixed powder was weighed out into a beaker and 250 ml deionised water was added and the mixture stirred. Solubilisation and reduction occurred concurrently to give a strongly coloured solution with an extinction coefficient at 400 nm, based on the molar concentration of silver present, of > 10,000 L mol ^"1 cm ^"1 .

Example 18

A solution of chitosan/ Ag ⁰ , prepared as described in Example 1 above, was spray dried by a conventional process and isolated as a fine, free-flowing brown powder that could be rapidly redissolved by addition of water to give a solution having similar λ _max and extinction coefficient values to those of the original solution.

Example 19

10 g of sodium carboxymethyl cellulose (DS = 0.9) was slurried in 200 ml Mopropyl alcohol containing 10% water while 0.3 g AgNO ₃ in 2.5 ml of deionised water was added drop wise. After stirring for 2 hours 0.5 g sodium ascorbate, dissolved in the minimum amount of water, was slowly run into the suspension. Stirring was continued for a further hour then the grey powder was filtered off, dispersed in 500 ml water, and the mixture stirred until a homogeneous solution was obtained.

Example 20

A suspension of 10 g of finely ground chitosan particles was stirred in 70 ml deionised water while 0.6 g AgNO ₃ in 5 ml deionised water was added drop wise to it. Stirring was continued until the supernatant did not give any precipitate on addition to a solution of NaCl. The suspension was diluted to 470 ml with deionised water and 9 ml lactic acid (80%) was added to solubilise the chitosan/Ag ⁺ complex. Once the complex was dissolved 2.5 g solid cetyltrimethylammonium bromide was added to the well-stirred solution, followed by 0.62 g ascorbic acid in 20 ml deionised water. No change in the colour of the solution was observed and there was no evidence of the development of the intense brown colour characteristic of the chitosan/Ag ⁰ complex. The product was isolated as a pale yellow-brown solid on raising the pH of the solution to > 10 by addition of NaOH, filtering and rinsing well with water and drying. Redissolving the recovered solid in 0.1 M lactic acid gave a clear colourless solution that had an extinction coefficient < 100 L mol ^"1 cm ^"1 , based on the molar concentration of silver present. Clear, colourless transparent films could be cast from the solution, which remained stable on standing for 3 months in daylight.

Example 21

The turbidity of the solutions prepared by the process according to the first aspect of the invention was compared with the solutions prepared by the processes disclosed in EP-A- 1312262 and WO 02/15698 (using photochemical reduction)

by determining the ratio of the absorbance at X _m3x , (the wavelength at maximum absorbance) and the absorbance at a wavelength as far as possible from λ _max - In the latter measurement, absorbance should be at a minimum and any excess loss of transmittance may be ascribed to light scattering due to turbidity. Hence, for solutions measured at approximately similar absorbance values at Xm _3x , ₄ oo _nm the ratio λ _max , ₄ Q _{0 nm} / λ _{max, 65} o _nm decreases as turbidity increases. The results obtained were as follows:

The results show that the chitosan-silver solutions prepared by the methods disclosed in EP-A-1312262 and WO 02/15698 have greater turbidity compared to the solution prepared by the process of the present invention.

Example 22

Chitosan-silver complexes were produced according to the process of the invention and tested for their activity against MSRA using the Swiss SNV 195- 920 test method for biocidal activity.

The test showed no zone of inhibition and a complete kill underneath the test sample where the MRSA was in contact with the treated sample, thus demonstrating that there is no soluble, unreduced ionic silver remaining in the material after the reduction step, hence there can be no migration of anti-microbial activity away from the chitosan.

In contrast, the presence of unreduced, soluble silver ions in the chitosan-silver compositions prepared according to the processes disclosed in EP-A-1312262 and WO 02/15698 is demonstrated by the biological test results reported therein.

Specifically, all the products tested, except Example 7 of WO 02/15698, are reported to show a large zone of inhibition when assessed against JSdRSA using the Swiss SNV 195-920 test, confirming that all these products contain a considerable proportion of a soluble, uncomplexed silver component in the form of unreduced ionic silver compounds which can migrate away from the chitosan during use.

The reason for the absence of a zone of inhibition in Example 7 of WO 02/15698 is due to the fact that the treated fabric is neutralized in aqueous ammonia, rinsed well and then dried prior to testing. In doing so, any ionic silver compounds present solubilise in the ammonia, thus allowing their removal prior to testing for biocidal activity. Accordingly, no zone of inhibition is observed.