WO2009043839A1

WO2009043839A1 - Coalescing carboxymethylchitosan-based materials

Info

Publication number: WO2009043839A1
Application number: PCT/EP2008/063046
Authority: WO
Inventors: Bryan Greener; Christopher Phillip Ledger
Original assignee: Smith & Nephew Plc
Priority date: 2007-09-29
Filing date: 2008-09-29
Publication date: 2009-04-09
Also published as: GB0719065D0

Abstract

A high molecular mass cationic polymer material has a first state which includes at least two separate but adjacent surfaces and a second state in which the polymer material consists of a homogeneous body. The material transitions from the first state to the second state upon hydration. Preferred polymer materials include carboxymethylchitosan. In a preferred preparation method, chitosan is mixed with a solution of base and chloroacetic acid in a mixture of water and alcohol, preferably ethanol.

Description

COALESCING CARBOXYMETHYLCHITOSAN-BASED MATERIALS

Field of the Invention

The invention relates to the field of absorbent, self-coalescing materials, in particular hydratable polymeric materials, and methods of preparing such materials. One application of such materials is to the field of wound dressings.

Background to Invention

Materials that swell upon absorption of solvent are well known and have been developed to enhance absorbency characteristics to generate super-absorbent materials. These materials have been applied in disposable nappies (diapers) and hydroponics for example. Commonly applied super-absorbent materials are based upon polymers with poly-acid functionality, such as salts of poly(acrylic acid). Super-absorbent materials are commonly presented in a granular physical form in the micrometer size range. This small particle size is important in enabling the super-absorbent properties of the material because larger monoliths suffer from the phenomenon of gel blocking, wherein solvent is rapidly absorbed within the surface of the object but this hydrated layer then impedes the hydration of the core of the particle, causing retarded hydration rates. The same phenomenon can also be manifest when attempting to swell bulk quantities of super- absorbent material in intimate contact; the surface particles fully hydrate and shield underlying particles from the solvent. Gel blocking is overcome in fluid absorbing applications by dispersing the particles in a significantly less absorbent (e.g. a wicking material) or non-absorbent matrix, commonly a non-woven material. In applications that require pre-swollen super-absorbent gel, the particles can be hydrated by slow addition to a large excess of liquid being stirred rapidly.

The physical format of super-absorbent materials was restricted to micrometer-size particles for commercial applications until recently, when Courtaulds Ltd (US5731083) generated carboxymethylcellulose-based super-absorbent fibres of micrometer-size diameter. These fibres were not the first super-absorbent fibres of their kind (see for example US4405324, Morca Inc.), but the precise processing conditions applied to first generate cellulose fibres and second to convert them to carboxymethylcellulose fibres was such that new combinations of physical properties were developed in the final product (e.g. mechanical strength and fluid absorbency). These new combinations of properties provided the basis for claims relating to the invention. Non-woven formats of this material of a few millimetres in depth were capable of rapid hydration to their full depth because liquid could penetrate the entire device prior to the onset of gel-blocking (this capability is related to the open volume of the non-woven material). This material was very successfully commercialised as a wound dressing (Aquacel, ConvaTec Ltd), exploiting its unique fluid handling properties.

In the intervening years since this invention, alternative technological solutions to these unique physical properties have not been discovered. However, in common with all technology, super-absorbent fibres generated from carboxymethylcellulose (CMC) can be improved upon in some respects for specific commercial applications. For example, when non-woven formats of CMC fibres are fully hydrated, the mechanical integrity of the hydrogel is relatively weak and the material can easily be disrupted under light pressure or be physically dissociated in excess solvent. A major contributor to this weakness is the very low self-cohesion of hydrated CMC fibres. Furthermore, the exposure of CMC-based materials to cations such as silver cations (commonly applied in antimicrobial applications) or calcium cations (commonly applied in haemostatic applications) results in a significant disruption of physical properties and significantly increases the dissolution of the material in aqueous media; this is undesirable for some applications. In attempting to overcome these weaknesses, we generated materials that exhibited unexpected and surprising transformational properties, with potential for unique applications in a range of commercial sectors including medical applications.

This invention is exemplified by materials based upon the naturally occurring polysaccharide chitosan; more specifically, carboxymethylchitosan (CMCh). In common with the prior art that existed concerning the preparation of CMC and CMC fibres before Courtaulds made their invention, there exists prior art in the production of water-soluble CMCh and water-absorbent CMCh powders (see for example US3879376, L'Oreal) and water-absorbent CMCh fibres (see for example US4651725, Unitika Ltd). The effect on the water-absorption characteristics of chitosan resulting from conversion, by carboxymethylation, to CMCh are also well known (see for example Y. Qin et al, J. Appl. Polym. Sci., 2006, 99, 31 10). However, the physical properties of all of the water- soluble or water-absorbent chitosan derivatives disclosed in the prior art either result in materials that dissociate and dissolve in water or remain discrete, but swollen, upon water absorption. These properties limit the versatility of this material.

US 2002/0147318 describes a process for preparing a water-soluble carboxymethyl chitosan in which deacetylated chitosan is suspended in isopropyl alcohol and then treated with sodium hydroxide. Monochloroacetic acid is added. After 6 hours at 50⁰C, the mixture is poured into water, the pH is adjusted with glacial acetic acid and the resulting precipitate is collected, washed with methanol and dried. The resulting water soluble product has a molecular weight of 220 to 25OkDa.

Summary of Invention

The subject of this invention is a suitable composition that enables a new physical transformation. The physical transformation in question involves the conversion of a first stable physical geometry into a second stable physical geometry upon hydration, wherein hydration enables the self-coalescence (fusion) of spatially separated elements or surfaces of the first stable physical geometry. Paradigms for this process are shown below in Figure 1.

Each geometry is physically stable. Thus, immersion of the first stable physical geometry in excess solution results in conversion to the second stable physical geometry without significant loss of the material mass by dissolution. That is, the second stable geometry is insoluble, or has only very limited solubility, in the excess solution. The second stable physical geometry is, at least substantially, self supporting such that it is able to retain its shape when is excess solution, or when removed therefrom. In typical preferred forms in the second stable physical geometry the material of the invention is a gel or gel like material.

A feature of the invention is the physical homogeneity of the object in both the first and second physical geometries.

The novel transformation that is the subject of this invention is enabled by construction of the object, at least in part, from materials that can exist in physically stable forms in the dry state and the hydrated state. Furthermore, the hydrated state of the material must be sufficiently self-cohesive, even when immersed in excess solvent, to enable fusion to occur. This, we believe, is a property unique to a limited range of states of matter, some of which we prepare to exemplify this invention.

In broad terms the present invention relates to a composition of matter that, when formed into an object of suitable geometry, can self-coalesce upon hydration in a suitable solvent. According to a first aspect of the invention there is provided a high molecular mass cationic polymer material having a first state which includes at least two separate but adjacent surfaces and a second state in which the polymer consists of a homogeneous body, wherein the material transitions from the first state to the second state upon hydration.

Thus, on hydration the material expands and the surfaces merge or coalesce to result in a body of self supporting material, typically a gel or gel-like material, which has uniform properties in any dimension. Surfaces and other boundaries within the body of material are absent. Furthermore the body of material is insoluble, or at least of limited solubility in the hydrating solvent and is able to retain its physical geometry under leading (for example gravity).

The term 'suitable geometry' is taken to describe an arrangement where separate (for example spatially separate, but not necessarily physically separate) elements or surfaces of the object are sufficiently proximate to enable coalescence upon hydration- induced expansion.

The term 'suitable solvent' is taken to describe a fluid (liquid or gas) that can be absorbed be the object, causing expansion and a change in the physical properties of the object (e.g. surface energy). The suitable solvent is typically and preferably an aqueous medium.

The term 'self-coalesce' is taken to describe the transformation of two or more spatially separated physically homogeneous elements into a single physically homogeneous element (as in Figure 1 B), or of fusion of previously spatially separated surfaces of the same element (as in Figure 1 A).

Suitable compositions of matter from which objects of this invention can be formed are those comprised, entirely or in part, of high average molecular weight cationic polymers including zwitterionic (carrying both anionic and cationic charge) polymers with a cationic charge bias. The cationic polymer may be, or may be a derivative of, a synthetic or a naturally occurring polymer. Preferably, the cationic polymer is one carrying amine functionality. More preferably, the cationic polymer is a polysaccharide. More preferably still, the cationic polymer is chitosan or a derivative of chitosan. The chitosan may be derived from any source, marine or fungal, and is preferably of a weight average molecular weight (Mw) exceeding 10 kDa (kilodaltons), more preferably exceeding 100 kDa and most preferably exceeding 200 kDa.

Where the polymer is a derivative of chitosan, it is preferably a carboxylated derivative. More preferably, it is a carboxyalkyl derivative of chitosan. More preferably still, it is a carboxymethyl derivative of chitosan. The carboxymethyl derivative of chitosan is preferably of a weight average molecular weight exceeding 50 kDa, more preferably exceeding 100 kDa, especially exceeding 500 kDa, more especially exceeding 60OkDa and especially 70OkDa or more.

Carboxymethylation is preferably achieved using known reagents: a base and chloroacetic acid or preferably a neutral salt of chloroacetic acid such as sodium chloroacetate. Preferably, the reaction is carried out in a single step: chitosan fibres or (less preferably) particles being immersed in a solution of reagents or vice versa. Suitable reaction solvents include mixtures of an alcohol with water. The alcohol may be any known but is preferably a non-solvent for chitosan and carboxymethylchitosan, for example isopropanol. The base may be any known but is preferably a water-soluble inorganic base such as sodium hydroxide or potassium hydroxide, preferably sodium hydroxide.

According to a second aspect of the invention there is provided a method of preparing high molecular mass carboxymethylchitosan comprising the steps:

a. mixing chitosan with a solution of a base and chloroacetic acid, or a neutral salt thereof, dissolved in a reaction solvent comprising a mixture of an alcohol and water;

b. allowing the reaction to proceed at ambient temperature for at least 8 hours whilst ensuring adequate exposure of the chitosan to the reaction solvent;

c. when the reaction is complete, washing the reaction product in excess alcohol- containing solvent;

wherein the volume (in millilitres) of the reaction solvent is at least 20-times the mass (in grams) of chitosan. A high molecular mass carboxymethyl chitosan preferably comprises a carboxymethyl chitosan having a mass of at least 50OkDa, more especially at least 60OkDa and especially 70OkDa or more.

In one preferred embodiment the volume of reaction solvent (in millilitres) exceeds the mass of chitosan (in grams) by more than 20 but less than 70-times, more preferably by more than 30-times but less than 40-times.

In another preferred embodiment the mass of sodium chloroacetate exceeds the mass of chitosan by not more than 2-times, more preferably by not more that 1 .2-times.

In a preferred embodiment, the alcohol of the reaction solvent is isopropanol.

In further preferred embodiments the reaction is carried out at ambient temperature for a period of at least 8 hours, more preferably for at least 15 hours and even more preferably for at least 18 hours.

In a particularly preferred embodiment, the alcohol of the reaction solvent is isopropanol, the mass of sodium chloroacetate is not more than twice (more especially not more than 1 .2 times) the mass of the chitosan and the reaction is allowed to proceed for at least 8 hours.

When the chitosan is provided for reaction in powder or fibre form, this material should be adequately exposed to the turbid reaction solvent throughout the duration of the reaction. This process can be facilitated by any means known to the artisan but can be simply achieved by rolling the reaction vessel, for example.

When the reaction is complete, reaction by-products detrimental to the stability of the product, such as sodium chloride or sodium hydroxyacetate, should be removed to the maximum extent feasible. To achieve this, the reaction product is washed, preferably in one or more steps, in excess solvent comprised of at least 60 parts alcohol (such as ethanol) and 40 parts water (60:40).

More than one washing step is preferred and, when this is the case, the first wash step has preferably a higher water content than subsequent steps, with water content decreasing in every wash step. For example, a suitable two-step wash procedure involves a first wash in excess solvent comprised of at least 60 parts ethanol and 40 parts water (60:40) and a second wash in excess solvent comprised of at least 90 parts ethanol and 10 parts water (90:10).

Thus in a preferred embodiment the reaction product is washed in a plurality of washing stages, each employing an excess of a solvent comprising alcohol and water, wherein in each succeeding stage the solvent consists of a higher proportion of alcohol. Preferably the alcohol is ethanol

It is essential that wash solvents always includes some water to avoid excessive dehydration of the product, which can result in brittleness.

The composition of the wash solvent may include any suitable alcohols such as ethanol, isopropanol or methanol. Ethanol is preferred.

The product resulting from washing and solvent removal can be sterilised by methods typical for the sterilisation of medical devices, for example gamma-irradiation, electron- beam irradiation or ethylene oxide treatment.

Prior to radiation-based sterilisation, the washed reaction product should be adequately solvent-free. This can be achieved by any drying process known to the skilled artisan. A preferred drying process is conducted at temperatures not exceeding 40⁰C, more preferably not exceeding 30⁰C. Preferably, solvent removal is achieved by placing the material under a sub-atmospheric pressure. The pressure is preferably less than 500 mbar, more preferably less than 1000 mbar. The duration of the drying process, when achieved by vacuum drying, preferably exceeds 8 hours, more preferably exceeding 12 hours.

The weight average molecular weight of the material following washing and radiation sterilisation is preferably greater than 12OkDa, more preferably greater than 13OkDa and after washing and ethylene oxide sterilisation is preferably greater than 40OkDa, more preferably greater than 50OkDa. It is important that these molecular weights are obtained to avoid mechanical integrity problems in the final product and dissolution problems when exposed to fluid. Additives and co-components can be added at any stage of the above process, prior to terminal sterilisation. These agents may be any suitable for a topical or internal medical application, such as analgesics, anaesthetics, antimicrobial agents, anti-cancer agents, nicotine or nicotine substitutes or other synthetic or naturally-derived pharmaceuticals including peptides, proteins such as growth factors or retardants, enzymes (e.g. those facilitating tissue debridement), DNA or RNA fragments.

When the additive is an antimicrobial agent, it may be for example: silver or silver compounds, iodine or iodine compounds, quaternary amine-based antimicrobials such as polyhexamethylenebiguanide or chlorhexidene, antibiotics such as gentamycin, vancomycin or a peptide-based agent.

When silver is introduced into the formulation, and the formulation is carboxymethylchitosan-based, addition is preferably achieved by immersion in a solvent mixture of a similar composition as that applied during the carboxymethylation process.

In a third aspect, the invention provides a method of fusing two or more solid surfaces, wherein the surfaces are initially separate (in particular, spatially separated) but adjacent surfaces of one or more object(s) comprising a self-coalescing material as herein described, notably the high molecular mass polymer material of the first aspect of the invention. The method comprises the step of immersing said surfaces in an aqueous medium and thus hydrating and expanding the self-coalescing material. In one embodiment, the surfaces are initially spatially separated surfaces of the same object. Alternatively, the surfaces are initially spatially separated surfaces of different objects. These alternatives are not mutually exclusive. The surfaces may be the surfaces of fibres, for example in a woven or, more especially, a non-woven fibrous material. In such materials, the surfaces may have portions which are spaced apart and portions which, while being separate, are in contact.

Objects fabricated from the compositions defined above, and suitable for the method, need to be suitably designed to enable coalescence upon hydration. For example, an isolated linear object would not have the opportunity to self-coalesce upon hydration. In contrast, a pair of isolated but adjacent linear objects would have the opportunity to swell and coalesce upon hydration. In this context, 'adjacent' means located within about 10 mm of one another. Thus, suitable objects can be defined as containing, at least in part, spatially separated elements or surfaces located within about 10 mm of one another. Preferably, the spatially separated elements or surfaces are located within 5 mm of one another. More preferably, the spatially separated elements or surfaces are located within 1 mm of one another. In some cases, for example fibre based materials, at least parts of adjacent surfaces may be in contact.

Preferred physical formats that meet the above description are fibre-based materials such as woven and non-woven materials. Other suitable formats include knits, open- celled foams and laminates including corrugated materials. More complex arrangements can be fabricated by methods known to one skilled in the art, such as lithography, micromachining and electrospinning. The invention is not restricted to formats of high open area but includes solid monoliths. Fibre based materials are preferred and fibre-based non-woven materials are particularly preferred.

The invention is not restricted to objects consisting exclusively of self-coalescent material, but includes composites, for example composites of common medical device formats and self-coalescent material and surface-coatings, for example implantable metal- or biomaterial based devices including soft-tissue substitutes and joint implants. Composites suitable for topical and internal wound management include those combining polyurethane based materials, such as foams, slabs and films with self- coalescent materials, for example in powdered or, more especially, fibrous form.

When devices comprised, at least in part, of the compositions of the first aspect of this invention are immersed in a fluid, they absorb fluid, become swollen and self-coalesce across contact points. This invention is not restricted to specific compositions or specific fluids, but in preferred forms and for preferred end-sues, the fluid is most preferably water based. For example, in the case of carboxymethylchitosan-based materials, the fluid is preferably water based. Examples of water based fluids include water or a solution of water, such as saline or a biologically-derived fluid such as whole blood, blood plasma, serum, saliva, wound exudate or bone marrow aspirate.

Further aspects of this invention relate to the application of objects of the first aspect of this invention. The novel material properties of the described self-coalescing materials can be exploited in a range of applications, for example in irreversible fluid valving systems and moulding materials. However, the main purpose of this invention is directed at medical applications, for which the physical properties of the materials described are particularly suitable. Medical applications include, but are not restricted to, wound dressings, haemostatic dressings, void-fillers for open cavities and anti-adhesion sheets for the separation of internal organs. These applications encompass the use of the product in a dry or pre- hydrated state. For example, for application to burns or for the debridement of wounds, the application of a hydrated device is desirable. Alternatively, the dry device can be applied to the site of application and hydrated in situ, for example with saline solution or distilled water. Alternative medical uses of this invention are those involving the temporary concentration and localisation of chemical or biological entities, such as cells, bacteria, proteins or antibodies, within a hydrated matrix. Such an arrangement can be achieved by immersing the dry material in a solution of the chemical or biological entity or by applying a liquid containing the chemical or biological entity to the device. In this manner, a method of generating biologically active matrices can be obtained. Such matrices can be applied in hard and soft tissue defect repair.

A specific medical application of this invention is as a device for the generation of haemostasis at sites of arterial or venous rupture. These injuries may be the result of traumatic accident or battlefield wound. For the management of heavily bleeding wounds of large open volume, for example projectile exit wounds, self-coalescing carboxymethylchitosan can be provided in easy to apply fibrous form, for example as non-woven balled devices. Such devices are easily packed into wounds of large open volume. For the management of heavily bleeding wounds of low open volume, for example projectile entry wounds, self -coalescing carboxymethylchitosan can be provided in easy to apply tubular form, for example as a cylindrical 'tampon'. Such devices are easily inserted into wounds of low open volume.

Thus a further aspect of the invention provides a wound dressing, in particular for promoting or achieving haemostasis at a site of arterial or venous rupture, the dressing comprising a material of the first aspect of the invention in the form of a fibrous substrate, in particular a non-woven fibrous substrate. Most preferably the material is a carboxymethyl chitosan. More especially, the material is a carboxymethyl chitosan having a molecular mass of at 50OkDa or more (prior to sterilisation). Preferably the material has a molecular mass of at least 10OkDa after radiation sterilisation, preferably at least 12OkDa and more especially at least 13OkDa after radiation sterilisation, or, at least 30OkDa, more especially at least 40OkDa and in particular at least 50OkDa after ethylene oxide sterilisation. A further specific medical application of this invention is as a device for the prevention of tissue adhesions, particularly internal tissue adhesions between neighbouring organs or neighbouring parts of the same organ or between internal organs and the components of the skin. Such adhesions can cause significant discomfort, impairment of function and specific medical complications that require surgical intervention. For the avoidance of such adhesions, a soft, conformable and flexible non-woven sheet of carboxymethyl- chitosan can be provided, preferably in the weight per unit area range of 50-75 grrϊ². The dry non-woven material can be applied to tissues at risk of subsequent adhesion during a surgical procedure, the moist tissue providing a source of hydrating fluid for the applied non-woven sheet. The sheet becomes rapidly hydrated and self-coalesces to form a continuous barrier between the tissues.

The invention is described in more detail with reference to the following Figures in which:

Figure 1 shows paradigms of the conversion of a first stable physical geometry into a second stable physical geometry upon hydration, wherein hydration enables the self- coalescence (fusion) of spatially separated elements of the first stable physical geometry. In case A, the self-coalescence comprises the fusion of spatially separated surfaces of a single element; in case B the surfaces are adjacent surfaces of two separate elements;

Figure 2 shows a plug of material formed by the self-coalescence of carboxymethylchitosan fibres. The material demonstrates transparency, homogeneity and physical stability (see Example 7);

Figure 3 is a clot formed following immersion of carboxymethylchitosan fibres in excess whole human blood (see Example 9);

Figure 4 shows an image of carboxymethylchitosan-based non-woven material, following part-immersion in serum for 3 days (see Example 12);

Figure 5 shows, for the ex-vivo model of Example 16 employing a dressing using the material of the invention, and for two proprietary dressings, the rate at which blood is collected downstream from a wound as a function of time, and as a function of whether or not pressure is being applied to the dressing; and Figure 6 shows the absorption of biological fluid (serum or blood) by a material of the invention.

Examples

Example 1

Generation of self-coalescing carboxymethylchitosan fibres

A) Synthesis

Immediately prior to reaction, sodium chloroacetate (1 .75 g) was dissolved in 4% aqueous sodium hydroxide solution (7 ml). This solution was added to isopropanol (45 ml) and shaken vigorously, resulting in a turbid suspension. This mixture was added to a vessel containing chitosan fibres (1 .50 g), the container sealed and rolled at approximately 60 rpm for 18 hours.

B) Washing Steps

B1 ) After step A, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 99:1 ethanol:water (200ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight.

B2) After step A, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 60:40 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and transferred to a second vessel containing 90:10 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight. Example 2

Generation of self-coalescing carboxymethylchitosan fibres (scale-up)

Immediately prior to reaction, sodium chloroacetate (96.8 g) was dissolved in 4% aqueous sodium hydroxide solution (387 ml). This solution was added to isopropanol (2490 ml) and shaken vigorously, resulting in a turbid suspension. This mixture was added to a vessel containing chitosan fibres (83.0 g), the container sealed and rolled at approximately 60 rpm for 18 hours. After this time, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 99:1 ethanol:water (2000 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight.

Example 3

Radiation sterilisation of self-coalescing carboxymethylchitosan fibres

The material resulting from Example 1 , step B2 was packaged in gas-permeable sterilisation pouches and sterilised by gamma irradiation at 30-40 kGy. The molecular weight of the material pre-and post-sterilisation was determined by gel permeation chromatography. The molecular weight prior to sterilisation was approximately Mw 70OkDa; the molecular weight post-sterilisation was approximately Mw 14OkDa. The molecular weight change in the material, although substantial, was such that the physical properties of the material were not significantly altered by sterilisation.

Example 4

Ethylene oxide sterilisation of self-coalescing carboxymethylchitosan fibres

The material resulting from Example 1 , step B2 was packaged in gas-permeable sterilisation pouches and sterilised by ethylene oxide treatment. The molecular weight of the material pre-and post-sterilisation was determined by gel permeation chromatography. The molecular weight prior to sterilisation was approximately Mw 70OkDa; the molecular weight post-sterilisation was approximately Mw 575kDa. The molecular weight change in the material was such that the physical properties of the material were not significantly altered by sterilisation.

Example 5

Water absorbency of self-coalescing carboxymethylchitosan fibres

The material resulting from Example 3 (100 mg) was immersed in water (4 ml) for 1 minute and withdrawn. Excess liquid was allowed to drain and then the hydrated transparent mass was weighed. The material was calculated to absorb approximately 25-times its own mass in water without significant dissolution.

Example 6

Serum absorbency of self-coalescing carboxymethylchitosan fibres

The material resulting from Example 3 (100 mg) was immersed in serum (4 ml) for 1 minute and withdrawn. Excess liquid was allowed to drain and then the hydrated transparent mass was weighed. The material was calculated to absorb approximately 13-times its own mass in serum without significant dissolution.

Example 7

Self-coalescence of carboxymethylchitosan fibres in water

The material resulting from Example 3 (100 mg) was immersed in water (4 ml) for 1 minute and withdrawn. Excess liquid was allowed to drain and then the hydrated transparent mass was allowed to stand for 4 hours. After this time, the individual fibres of the material had self-coalesced and the material was then effectively a homogeneous, elastic hydrogel, able to stably retain its physical geometry under loading (Figure 2).

Example 8

Self-coalescence of carboxymethylchitosan fibres in serum The material resulting from Example 3 (100 mg) was immersed in serum (4 ml) for 1 minute and withdrawn. Excess liquid was allowed to drain and then the hydrated transparent mass was allowed to stand for 4 hours. After this time, the individual fibres of the material have self-coalesced and the material as effectively a homogeneous, elastic hydrogel, able to stably retain its physical geometry under loading.

Example 9

Blood-clotting ability of carboxymethylchitosan fibres

The material resulting from Example 3 (300 mg) was immersed in heparin-stabilised whole human blood (10 ml). The material was withdrawn after 10 seconds immersion. Approximately 3 ml of the blood remained in the container and a large coagulated clot had formed intimately within and upon the material of the fibres (Figure 3).

Example 10

Generation of self-coalescing carboxymethylchitosan non-woven material

The method reported in Example 1 , step B2 was repeated using chitosan non-woven material with an area weight of 100-150 gπϊ².

Example 11

Radiation sterilisation of self-coalescing carboxymethylchitosan non-woven material

The material resulting from Example 10 was packaged in gas-permeable sterilisation pouches and sterilised by gamma irradiation at 30-40 kGy. The molecular weight of the material pre-and post-sterilisation was determined by gel permeation chromatography. The molecular weight prior to sterilisation was approximately Mw 70OkDa; the molecular weight post-sterilisation was approximately Mw 14OkDa. The molecular weight change in the material, although substantial, was such that the physical properties of the material were not significantly altered by sterilisation.

Example 12 Device stability in excess fluid

The material resulting from Example 1 1 was cut to a 10 x 2.5 cm strip and was partly immersed, at one end, in serum (5 ml) for 3 days. After this period, the serum-immersed section of the device was swollen, transparent, homogeneous and integral and could easily be withdrawn by the dry section of the strip (Figure 4).

Example 13

Generation of silver-ion loaded carboxymethylchitosan non-woven device

This process was carried out in subdued lighting conditions.

The carboxymethylchitosan non-woven substrate produced in Example 10 (1 .5 g, approximately 1 O x 10 cm) was immersed in a pre-prepared solution of silver nitrate (70 mg) dissolved in distilled water (7 ml) and isopropanol (45 ml). The vessel containing the non-woven and reactants was rolled for 18 hours. After this time, the non-woven was removed and immersed in 99:1 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight and stored in the absence of light.

Example 14

Radiation sterilisation of self-coalescing silver carboxymethylchitosan fibres

The material resulting from Example 13 was packaged in gas-permeable sterilisation pouches and sterilised by gamma irradiation at 30-40 kGy. The molecular weight of the material pre-and post-sterilisation was determined by gel permeation chromatography. The molecular weight prior to sterilisation was approximately Mw 70OkDa; the molecular weight post-sterilisation was approximately Mw 14OkDa. The molecular weight change in the material, although substantial, was such that the physical properties of the material were not significantly altered by sterilisation.

Example 15 Demonstration of physical property matching in silver and non-silver carboxymethylchitosan-based devices in contrast to carboxymethylcellulose devices

The non-woven materials produced in Example 1 1 (non-silver) and Example 14 (silver) were compared with a set of non-silver (Aquacel, ConvaTec Ltd) and silver (Aquacel Ag, ConvaTec Ltd) carboxymethylcellulose-based non-woven devices of similar weight per unit area and physical format. The materials were very similar in appearance when dry. 2.5 x 10 cm strips of each material were partly immersed, at one end, in distilled water for a period of 30 seconds. After this time, samples were withdrawn and physically evaluated. Both of the silver-impregnated samples were significantly less transparent that their respective non-silvered counterparts, both of which were transparent in appearance. Fluid absorbencies were measured to be very similar for all samples.

The carboxymethylchitosan-based samples were indistinguishable to the touch and the wet section was swollen, homogeneous and integral and could easily be withdrawn by the dry section of the strip. In contrast, the carboxymethylcellulose-based samples were grossly dissimilar: the non-silver sample was similar to both carboxymethylchitosan materials whereas the silver sample's wet section was of a significantly lower viscosity and integrity than any other material.

This example demonstrates that addition of cations, such as silver ions, to negatively charged superabsorbers, such as carboxymethylcellulose, has a detrimental effect on their solution-phase integrity. This effect is likely to be due to the cations forming ion- pairs with the carboxylate groups and increasing the degree of dissolution of the material. The inventors suggest that the same effect does not occur to such a significant extent in carboxymethylchitosan-based material because these are positively charged superabsorbers, with a minority of carboxylate groups compared to amine groups. The addition of small quantities of additional cations, such as silver, does not significantly alter the anion-cation balance of the polymer, in contrast to the case with carboxymethylcellulose. In addition, it is also likely that non-ion pair binding of silver ions can occur in carboxymethylchitosan via electron donation from uncharged amine groups. This is not a possibility in carboxymethylcellulose.

Example 16

Haemostat performance: Management of moderate to severe bleeding The fibres from Example 1 , step B2 were processed into a non-woven sheet of density 0.06-0.08 g/cm³, and sterilised as described in Example 3. A 10x10 cm pad from the resulting sheet was used to demonstrate the ability to manage moderate to large quantities of blood in an ex-vivo model.

The ex-vivo model consists of a 60 ml syringe filled with porcine blood treated with sodium citrate (10% v/v anticoagulant), attached to a piece of silicone tubing (5 mm diameter), which is passed through the muscle layer of a piece of porcine abdomen, and opens onto a beaker placed on a balance. The porcine abdomen is cut on a 5 cm length above the tract of the tubing and in the same direction. Muscle tissue is cut in depth until the tubing is reached, creating a model wound which is typically 5 cm long, 2.5 cm deep, and 2 cm wide. The syringe is placed in a syringe pump, and as the delivery of blood starts, a 3 mm biopsy punch is used to puncture the tubing within the wound. As blood leaks into the wound, the amount accumulated in the beaker downstream is reduced, until treatment (dressing) is applied: this intervention reduces the bleeding in the wound, and increases the flow of blood in the tubing downstream of the wound and into the beaker. In addition to the dressing using the material of the invention, two proprietary dressings were tested, that is, dressings available under the trade names QuickClot and HemCon

Figure 5 shows the rate at which blood is collected downstream of the wound into the weighed beaker, as a function of time, and as a function of whether pressure is being applied to the dressing or not. Full-shaded markers (e.g. •) denote application of manual pressure on the treatment in the wound, and whited-out markers (e.g. o) denote no applied pressure. The results shown in Figure 5 as "INV" ( ■ and D ) relate to the material of the invention (per Example 1 , as noted above).

The baseline flow rate measured with intact tubing is also plotted, and its variability marked by a grey zone centred on the average baseline.

Figure 5 demonstrates that of the 3 dressings tested, only the dressing using the material of the invention allowed the bleeding in the wound to be managed after removal of pressure at 1 minute: this dressing allowed a baseline-like blood flow to be retained downstream from the treated wound. In addition, after completion of the experiment, removal of the dressing using the materials of the invention was non-traumatic to the tissue, as the coherent gel formed did not adhere to the edges of the wound.

Example 17

Haemostat performance: Rate of absorbency of blood and serum

The fibres from Example 1 , step B2 were processed into a non-woven sheet of density 0.06-0.08 g/cm³. Squares of 2cm by 2 cm were cut out (approximately 50 mg each), and immersed for different periods of time in vials containing 15 ml of either citrated porcine blood or foetal calf serum, at room temperature.

The samples were weighed dry before immersion, and wet and swollen after removal from the vial and draining of excess fluid against the wall of the vial. The fluid uptake was measured as the relative proportion of fluid retained compared to the dry mass of the sample.

The time points chosen were 30 seconds, 1 minute, 2 minutes, 5 minutes, 10 minutes, and 4 hours. This time course was chosen to demonstrate the immediate absorbency power of the material of Example 2 when processed in a non-woven format.

Figure 6 shows that the material absorbs around 20 times its dry mass of biological fluid (serum or blood) within 30 seconds. In the case of blood, saturation in the small sample used (2x2 cm) seems to be reached after around 5 minutes.

This rapid absorption of blood, which occurs at room temperature and without any thermal effect, is useful in the performance of a haemostat: red blood cells, naturally activated platelets and clotting factors are concentrated due to the absorption of fluid by the device, creating a more favourable environment for natural haemostasis.

Comparative Examples

Example A

Generation of non-coalescing carboxymethylchitosan fibres Immediately prior to reaction, sodium chloroacetate (3.00 g) was dissolved in 4% aqueous sodium hydroxide solution (7 ml). This solution was added to isopropanol (45 ml) and shaken vigorously, resulting in a turbid suspension. This mixture was added to a vessel containing chitosan fibres (1 .50 g), the container sealed and rolled at approximately 60 rpm for 18 hours. After this time, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 99:1 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight. The resulting material was brittle and had a visible coating of a crystalline material. This material was sodium chloroacetate derived.

This example demonstrates that reactions utilising an excessive amount of sodium chloroacetate produce a final material from which impurities cannot be sufficiently removed to enable desired mechanical properties in the product.

Example B

Generation of non-coalescing carboxymethylchitosan fibres

Immediately prior to reaction, sodium chloroacetate (1 .75 g) was dissolved in 0.4% aqueous sodium hydroxide solution (7 ml). This solution was added to isopropanol (45 ml) and shaken vigorously, resulting in a turbid suspension. This mixture was added to a vessel containing chitosan fibres (1 .50 g), the container sealed and rolled at approximately 60 rpm for 18 hours. After this time, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 99:1 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight. The resulting material did not absorb more than 6-times its own weight in water and the majority of the absorbed liquid was not absorbed into the fibres but held in between the fibres.

This example demonstrates that reactions utilising an insufficient quantity of sodium hydroxide produce an insufficiently absorbent final product. A sufficient quantity of sodium hydroxide is required to activate the reactive sites of the chitosan (both alcohol and amine).

Example C

Generation of non-coalescing carboxymethylchitosan fibres

Immediately prior to reaction, sodium chloroacetate (1 .75 g) was dissolved in 4% aqueous sodium hydroxide solution (7 ml). This solution was added to ethanol (45 ml) and shaken vigorously, resulting in a turbid suspension. This mixture was added to a vessel containing chitosan fibres (1 .50 g), the container sealed and rolled at approximately 60 rpm for 18 hours. After this time, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 99:1 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight. The resulting material was as mechanically strong and absorbent as the material produced in Example 1 but was non-coalescent; fibres absorbed liquid but did not form a cohesive mass of uniform density on standing (c.f. Example 7).

Example D

Generation of non-coalescing carboxymethylchitosan fibres

Immediately prior to reaction, sodium chloroacetate (1 .75 g) was dissolved in 4% aqueous sodium hydroxide solution (7 ml). This solution was added to methanol (45 ml) and shaken vigorously, resulting in a turbid suspension. This mixture was added to a vessel containing chitosan fibres (1 .50 g), the container sealed and rolled at approximately 60 rpm for 18 hours. After this time, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 99:1 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight. The resulting material was as mechanically strong and absorbent as the material produced in Example 1 but was non-coalescent; fibres absorbed liquid but did not form a cohesive mass of uniform density on standing (c.f. Example 7).

Examples C and D demonstrate that using ethanol or methanol as the reaction solvent, results in an absorbent but non-cohesive product.

Example E

Generation of non-coalescing carboxymethylchitosan fibres

Immediately prior to reaction, sodium chloroacetate (1 .75 g) was dissolved in 4% aqueous sodium hydroxide solution (7 ml). This solution was added to ethanol (45 ml) and shaken vigorously, resulting in a turbid suspension. This mixture was added to a vessel containing chitosan fibres (1 .50 g), the container sealed and rolled at approximately 60 rpm for 4 hours. After this time, the fibres were removed from the now clear reaction solvent and transferred to a vessel containing 99:1 ethanol:water (200 ml). The material was disturbed every 15 minutes for 1 hour, after which time the material was removed and physically dried by the application of hand pressure between several layers of absorbent material. Following gross drying, the material was vacuum dried at ambient temperature overnight. The resulting material did not absorb more than 6-times its own weight in water and the majority of the absorbed liquid was not absorbed into the fibres but held in between the fibres.

This example demonstrates that reaction times less than 8 hours in duration are insufficiently long to allow sufficient carboxymethylation for the desired absorbency properties to be manifest in the final product.

Claims

1 . A high molecular mass cationic polymer material having a first state which includes at least two separate but adjacent surfaces and a second state in which the polymer consists of a homogeneous body, wherein the material transitions from the first state to the second state upon hydration.

2. A material according to claim 1 , wherein the cationic polymer is a polysaccharide.

3. A material according to either claim 1 or claim 2, wherein the cationic polymer is one carrying amine functionality.

4. A material according to claim 1 , 2 or 3 wherein the cationic polymer is chitosan or a derivative of chitosan.

5. A material according to claim 4 where the polymer is a carboxymethyl derivative of chitosan.

6. A material according to any preceding claim wherein the weight average molecular weight (Mw) of the material is 10OkDa or greater.

7. A material according to either claim 5 or claim 6, wherein the weight average molecular weight (Mw) of the material is 50OkDa or greater.

8. A method of preparing high molecular mass carboxymethylchitosan comprising the steps:

b. allowing the reaction to proceed at ambient temperature for at least 8 hours whilst ensuring adequate exposure of the chitosan to the reaction solvent; c. when the reaction is complete, washing the reaction product in excess alcohol-containing solvent;

wherein said method is characterised in that: the volume (in millilitres) of the reaction solvent is at least 20-times the mass (in grams) of chitosan.

9. A method according to claim 8, wherein the chloroacetic acid or neutral salt thereof is sodium chloroacetate and further wherein the mass of sodium chloroacetate in the reaction is not more than twice the mass of chitosan.

10. A method according to claim 9, wherein the mass of sodium chloroacetate in the reaction is not more than 1 .2-times the mass of chitosan.

1 1 . A method as claimed in claim 8, 9 or 10 wherein the alcohol of the reaction solvent is isopropanol.

12. A method according to any of claims 8 to 1 1 , wherein the reaction is allowed to proceed for at least 18 hours.

13. A method according to any of claims 8 to 12, wherein the reaction product is washed in an excess of a solvent comprising alcohol and water in a ratio in the range of 60:40 to 99:1 .

14. A method according to any of claims 8 to 13 wherein the reaction product is washed in a plurality of washing stages, in an excess of a solvent comprising alcohol and water, wherein in each succeeding stage the solvent consists of a higher proportion of alcohol.

15. A method of any of claims 8 to 14 further comprising a drying step comprising drying at a temperature not exceeding 40O.

16. A method according to claim 15, comprising drying at a temperature not exceeding 30⁰C.

17. A method according to either claim 15 or claim 16, wherein the drying step is conducted at a pressure of less than l OOOmbar.

18. A method according to claim 17, wherein the drying step is conducted at a pressure of less than 500mbar.

19. A method according to any of steps 8 to 18, further comprising a sterilisation step comprising exposing the dried reaction product to a sterilising agent selected from gamma radiation, electron beam radiation and ethylene oxide.

20. A method according to any of steps 8 to 19, further comprising the addition of an antimicrobial agent, analgesic agent, anaesthetic agent, anti-cancer agent, enzyme, enzyme-inhibitor, peptide, protein, DNA fragment or RNA fragment.

21 . A method according to claim 20, wherein the antimicrobial agent is selected from the list consisting of silver, a silver compound, iodine, an iodine compound, a quaternary amine-based compound, polyhexamethylenebiguanide, chlorhexidine, an antibiotic, gentamycin, vancomycin, and a peptide based agent.

22. A method according to claim 21 , wherein the antimicrobial agent is silver and further wherein silver is added to the reaction solvent prior to or during the reaction.

23. A self-coalescing material comprising a high molecular mass carboxymethylchitosan produced by the method of any of claims 8 to 22.

24. A self-coalescing material according to any of claims 23, in the form of a fibre-based material.

25. A material as claimed in any of claims 1 to 7 wherein in the first state the material is a fibre-based material, the separate but adjacent surfaces being surfaces of the fibres.

26. A self-coalescing material according to claim 24 or 25 in the form of a woven, knitted or non-woven material.

27. A self-coalescing material according to any of claims 1 to 7, or 23, in the form of an open-celled foam.

28. A self-coalescing material according to any of claims 1 to 7, or 23, in the form of laminated material.

29. A method of fusing two or more solid surfaces, wherein the surfaces are initially spatially separated but adjacent surfaces of one or more object(s) comprising a material according to any of claims 1 to 7, or 23 to 28, said method comprising the step of immersing said surfaces in an aqueous medium and thus hydrating and expanding the material.

30. A method according to claim 29 wherein the surfaces are initially spatially separated surfaces of the same object.

31 . A method according to claim 29 wherein the surfaces are initially spatially separated surfaces of different objects.

32. A wound dressing comprising a material of any of claims 1 to 7.

33. A wound dressing comprising a material of any of claims 23 to 28.

34. A wound dressing comprising the material according to claim 25 in the form of a non-woven woven substrate.

35. A wound dressing as claimed in claim 34 wherein the high molecular mass cationic polymer material is a carboxymethyl chitosan having a molecular weight of at least 50OkDa.

36. A wound dressing according to any of claims 32 or 35, wherein said wound dressing is a haemostatic dressing.

37. A haemostatic dressing according to claim 36, wherein the dressing comprises a non-woven material in a balled or cylindrical form.

38. A wound dressing according to claim 32, wherein said wound dressing is a void- filling dressing.

39. A wound dressing according to claim 32, wherein said wound dressing is an anti- adhesion sheet.

40. A self-coalescing material comprising a high molecular mass cationic polymer.

41 . A self-coalescing material as claimed in claim 40 wherein said high molecular mass cationic polymer is a high molecular mass cationic polymer material having any one or any permitted combination of the features defined in any of claims 1 to 7 or 23 to 28.