WO2008010199A2 - A nanofibre product - Google Patents

Abstract

A mesh comprises nanofibers of oxidised polysaccharide and a fibre-forming polymer. The oxidised polysaccharide is uniformly dispersed in the form of molecules and/or nanoparticles in a matrix of the fibre-forming polymer. The oxidised polysaccharide may be polyanhydroglucuronic acid or salts or intermolecular complexes thereof. The polymer may be PVA. The mesh may be used in wound dressings. The dressing may have multiple layers.

Description

"A nanofibre product"

Introduction

The invention relates to a nanofibre product. In particular the invention relates to a nanofibre product comprising an oxidised polysaccharide.

Nanofibres are fibres with a diameter of 1,000 nm or less. Nanofibres are typically formed from natural and synthetic polymers or polymer blends by an electrostatic spinning method.

WO 05/024101 discloses a method of producing nanofibres from a polymer solution using electrostatic spinning in an electric field created by a potential difference between a charged electrode and a counter electrode and a device for carrying out the method.

EP 1 591 569 discloses a process for the production of nanofibres with a diameter below 500nm in an electrostatic field.

Nanofibres have many applications and have been used in different technical fields such as filters for filtering gases and liquids.

US Patent Application No. 2004/0234571 discloses a process for injecting nanometer- scaled fibres directly into an intended body site of a patient. The process includes the steps of preparing a precursor fluid for the fibres and injecting the precursor fluid into the intended body site under the influence of an electrical field established between two electrodes to produce nanometer-scaled fibres for forming a reinforcement platform. A polymer is then optionally injected into the intended body site to form a nanofibre- polymer composite structure. The composite structure may contain interconnected macro pores wherein cells can grow and proliferate. The injected composite structure may also be used as a means of controlled drug release or bone reinforcement. However, such a method requires expensive equipment and may cause patient discomfort. Statements of Invention

According to the invention there is provided a mesh comprising nanofibers of oxidised polysaccharide and a fibre-forming polymer.

In one embodiment the oxidised polysaccharide is uniformly dispersed in the form of molecules and/or nanoparticles in a matrix of the fibre-forming polymer.

The oxidised polysaccharide may be oxidised glucan. The oxidised glucan may be polyanhydroglucuronic acid [PAGA] or salts or intermolecular complexes [IMCs] thereof. The salts may be derived from inorganic cations selected from the group Li⁺, Na⁺, K⁺, Ag⁺ , Mg²⁺Ca²⁺ , Zn²⁺, Co²⁺, Cu²⁺, Al³⁺, Fe²⁺,Fe³⁺, Ga³⁺, sodium and calcium.

In one embodiment the weight ratio of polysaccharide to fibre-forming polymer is from 99:1 to 20:80. Alternatively the ratio is from 80:20 to 60:40.

In one embodiment the fibre-forming polymer is substantially soluble in water. In this case the fibre-forming polymer soluble in water may be polyacrylic acid and/ or salts thereof and/or water soluble copolymers thereof, poly(met)acrylamide, polyhydroxypropylmethacrylamide, polyvinylalcohol. salts of chitosane, salts of alginic acid, cellulose derivatives soluble in water, or their mixtures, or polyvinyl alcohol.

In another embodiment the fibre-forming polymer is subtantially insoluble in water. In ^■ this case the copolymer may be polyurethane, polyacrylate, polyesters or cellulose derivatives that are insoluble in water.

In one embodiment the nanofibres have an average diameter in the range of from 50 nm to 1000 nm. preferably from 100 nm to 500 nm, most preferably from 200 nm to 400 nm. In one embodiment the mesh has a specific surface area in the range of from 1 m²/g to 100 m²/g. The specific surface area may be in the range of from 5 m²/g to 50 m²/g.

In one embodiment the mesh has an average pore size of from 10 nm to 400 nm, typically i from 20 nm to 80 nm.

In one embodiment the polyanhydroglucuronic acid [PAGA] or salt thereof may be derived from a starch, cellulose or gum, or may be of microbial origin for example polycellobiuronic acid.

I

The PAGA may comprise a microdispersed cellulose or derivative thereof. The PAGA or salt thereof may comprise a biocompatible salt thereof, a copolymer thereof or a biocompatible intermolecular complex (IMC) thereof.

The biocompatible intermolecular polymer complex may be a complex of:

an anionic component comprising polyanhydroglucuronic acid, which is partially or completely hydrolysed in a normal and/or oxidative environment for example a polyanhydroglucuronic acid containing material; and

a cationic component comprising an amino acid and biogenic amine thereof and/or antibiotics or A chemotherapeutic.

The biocompatible intermolecular polymer complexes may be complexes of PAGA with basic or uncharged polar amino acids such as histidine. arginine, glutamine. serine, lysine, and biogenic amines thereof such as histamine, guanidine. or ethanolamine. Alternatively the biocompatible intermolecular polymer complexes are complexes of PAGA with aminoglycoside antibiotic and/or aminopenicilins or amidinopenicilins such as polymyxins, bacitracin, neomycine, gentamicin.

Alternatively, the biocompatible intermolecular polymer complexes may be complexes of a chemotherapeutic with PAGA such as complexes of an antiseptic with PAGA. Antiseptic type cations in complexes with PAGA may be amino compounds such as the biguanid derivatives (e.g.chlorohexidine), heterocyclic amine ■ (e.g. hexetidine), quaternary ammonia cations of the type cetyltrimethylammonium, karbetopendecinium, benzethonium or cations belonging to the groups acridine (such as acridine derivatives, acriflavinium or ethacridinium cations); triphenylmethane (e.g. cations of fuchsoniminium derivatives such as methylrosanilinium); or phenothiazine dyes (such as cation of 3,7-tetramethyldiaminofenothiazinium).

In one embodiment the polyanhydroglucuronic acid or salt or IMC thereof may contain from 8 percent to 30 percent by weight of carboxyl groups. At least 80 percent by weight of the carboxyl groups may be uronic groups. At most 5 percent by weight of carboxyl groups may be carbonyl groups. The polyanhydroglucuronic acid may contain up to 0.2 percent by weight of bound nitrogen in their polymeric chain.

The molecular mass of the polymeric chain of the polyanhydroglucuronic acid and salt or IMC thereof may be from 1 kDaltons to 700 kDaltons, typically 5 kDaltons to 400 kDaltons.

In one embodiment the content of the carboxyl groups is in the range of from 18 percent by weight to 26 percent by weight of the polyanhydroglucurenic acid or salt orIMC thereof. At least 95 percent of these groups may be uronic groups. In one embodiment the polyanhydroglucuronic acid or salt thereof may contain at_. most 5 percent by weight of carbonyl groups. The carbonyl groups may be introduced into the PAGA molecule by a specific reaction of a periodic acid such as HIO₄ or salts thereof.

The nanofibres may be crosslinked. In one case crosslinking of the nanofibres may be created by covalent reactions between reactive groups of PAGA and PAGA or reactive groups of PAGA with reactive groups of fibre-forming polymers. The crosslinking of the nanofibres may be created by condensation reaction groups such as -COOH, -COOR (wherein R may be any suitable chemical entity), -OH₅ -NH₂, -CONH₂, -CONHNH₂₅ - CONHNHOC-, -CON₃. Typical catalysers of condensation crosslinking process are Lewis acids. The Lewis acids may be selected from the group of H₂SO₄, HCl, H₃PO₄, NaH₂PO₂, ZnCl₂, TiCl₄ or phosphorus oxides or chlorides

In one embodiment the nanofibres may contain a modifier and/or an emollient.

The modifier may be one or more selected from the group comprising: monomer or polymer acids, hydroxyacids, such as tartaric acid, citric acid, malic acid or malonic acid, succinic or maleic acid or their isomer fumaric acid or mixture thereof.

The emollient may one or more selected from the group comprising: polyhydroxycompounds and/or amino alcohols such as glycerol and polyglycerols, ethylene and propylene glycols and low molecular weight polymers and copolymers thereof (e.g. poloxamer 407) or aminoalcohols, e.g. mono-, di-, triethanol amine and mixture thereof.

The invention also provides a wound dressing comprising a mesh of the invention.

In another aspect the invention provides a biocompatible intermolecular polymer complex of: an anionic polysaccharide component and

a cationic component comprising an amino acid or biogenic amine thereof and/or antibiotics or chemotherapeutic.

The anionic component may comprise polyanhydroglucuronic acid containing material for example a polyanhydroglucuronic acid, which is partially or completely hydrolysed in a normal and/or oxidative environment.

In one embodiment the biocompatible intermolecular polymer complexes are complexes of PAGA with amino acids such as histidine, arginine, glutamine, serine, lysine, and biogenic amines thereof such as histamine, guanidine, or ethanolamine.

The biocompatible intermolecular polymer complexes may be complexes of PAGA with aminoglycosidic antibiotic and/or aminopenicilins or amidinopenicilins such as polymyxins, bacitracin, neomycine, gentamicin.

Alternatively the biocompatible intermolecular pofymer complexes of a chemotherapeutic and PAGA may be complexes of antiseptic and PAGA. The antiseptic type cations in complexes with PAGA may be amino compounds such as biguanid derivatives (e.g.chlorohexidine), heterocyclic amine (e.g. hexetidine), quaternary ammonia cations of the type cetyltrimethylammonium, karbetopendecinium, benzethonium or cations belonging in the groups acridine (such as acridine derivatives,, acriflavinium or ethacridinium cations), triphenylmethane (e.g. calions of fuchsoniminium derivatives such as methylrosanilmium); or phenothiazine dyes (such as cation of 3.7-tetramethyldiaminofenothiazinium).

The invention further provides a wound dressing comprising a complex of the invention. In another aspect the invention provides biocompatible, biodegradable and/or resorbable nanofibres comprising a matrix of random biocompatible polymers, this may include oxidised glucan with limited soluble or insoluble colloid dispersing particles of size 30nm-1000nrn and/or soluble oxidised glucan, with fibre-forming polymer mixable form, in amount of 1 % w/w-80% w/w nanofibres

The matrix of nanofibres may be manufactured using water soluble or water insoluble polymers or mixtures thereof.

The water soluble polymers may be one or more selected from the group comprising: poly-hydroxy substances and polyamino substances such as polyvinyl alcohols, polyethylenglycols, hydroxypropyl, hydroxypropylcarboxymetyl cellulose, methyl, ethyl cellulose, polyhydroxyethyl starch, amino ethyl cellulose, amino derivates of gluco or galacto marmans such as hydroxypropyltrimonnium chloride guargum, polyallylamine, polyethylenimines, poly(met)acrylamide, polyhydroxypropyl(rnet)acrylarnide,. polyvinylpyrrolidone, polyvinyl alcohol, chitosan, hydroxyethyl chitosan. hydroxyethyl alginate, then salts of polycarboxyl acids like polygalacturonic acid, hyaluronic or alginic acid, carboxymethyl cellulose and poly(met)acrylic acid and its copolymers with esters, also copolymers of maleinanhydride or fumaric acids with styrene or their terpolymers with acrylates and their mixtures.

In one embodiment the water soluble polymer is polyvinylalcohol (PVA) with a hydrolysis degree of 80%-98% and a molecular weight of from 8OkDa-12OkDa.

The water insoluble polymers may be selected from glucans such as cellulose and esters thereof (acetates, acetobutyrate or formiate), chitin, polylactic acid and glycolic acid copolyesters thereof, polyamides, e.g. polyamide 6,6. copolymers of maleinanhydride or fumaric acid with styrene or its terpolymers with acrylic acid and its esters, fibre forming polyurethane, poly(met)acrylic acid and its copolymers with their esters. In one embodiment the fibre forming system may contain emollient or modifying components.

The emollient substances contained in the nanofibres can be polyhydroxy compounds; amino alcohols, like glycerine and poly glycerol; ethylene; propylene glycols and low molecular weight polymers and copolymers thereof such as poloxamer 407; or amino alcohols such as mono, di, triethanolamine and its mixtures thereof.

The modifying substances may be incorporated into the polymer chain of a nanofibre by chemical reaction. For example by reacting low molecular weight substances with reactive groups that can undergo condensation reaction. The modifying substances may be selected from group of monomer acids; poly acids; or hydroxy acids, like tartaric acid, citric acid, malic acid or malonic acid, succinic acid and maleic acid or its isomer — fumaric acid, 1,2,3,4-butanetetracarboxylic acid, glycolic acid or lactic acid or its derivates and mixtures thereof.

The nanofibres of the mesh may not be crosslinked. Alternatively the invention also provides biocompatible, biodegradable and/or resorbable nanofibres which are formed by chemical crosslinking.

The chemical crosslinking reaction may be a condensation reaction with an acidic catalyst.

The condensing catalysts may be H₃PO₄, H₂SO₄ ,NaH₂PO₂ or mixtures thereof.

The oxidised glucan may be a microdispersed form of hydrolysed oxidised cellulose

The hydrolysed oxidised cellulose may be in the form of a soluble or partially soluble salts or intermolecular complexes of polyanhydroglucuronic acid. The cation of PAGA salts may be inorganic cation like Li⁺, Na⁺, K⁺, Ag⁺, Mg²⁺ Ca²⁺, Zn²⁺, Co²⁺, Cu²⁺, Al³⁺, Fe²⁺,Fe³⁺, Ga³⁺, or mixed salts thereof.

The organic cation of PAGA complex salts may be amino acids like histidine, arginine, glutamine, serine or lysine and their biogenic amines like histamine, guanidine, or colamine.

The organic cations of PAGA salts may be from the group of antimicrobial substances. The antimicrobial substances as a cation of PAGA salts are amino substances, like derivates of biguanide, hetero-cyclic amine (like hexetidine), quarter ammonic cations like cetyltrimethylamonium, carbetopendecinium, or benzethonium. The antimicrobial substances as a cation of PAGA salts may be from the acridine group for example derivates of acridine, acriflavinium or ethacridinium cations, or the trifenylmethane group for example cations of fuchsoniminium derivates like methylrosanilinium or fenothiazine dyes for example cation of 3,7-tetramethyldiamino-fenothiazinium.

The antimicrobial substances of a cation of PAGA salts or complex salts thereof may be from an antibiotic group. For example from the group of basic antibiotics of for example polypeptide type antibiotics such as bacitracin and polymyxines or aminoglycoside type antibiotics such as like neomycin, or gentamicin.

The invention further provides a product comprising a mesh as described herein. The product may comprise a number of layers, at least one of the layers comprising a mesh. The product may further comprise an agent to alter the flexibility of the mesh. The agent may increase the flexibility of the mesh alternatively, the agent may decrease the flexibility of the mesh. The agent may comprise a polymer selected from the group comprising polyethylene glycol (PEG); polyethylene oxide (PEO); polyurethane (PUR) and combinations thereof. The product may further comprise one or more additives. The additives may be selected from the group comprising: antimicrobials, antiseptics, antibacterials, antioxidants, vitamins, minerals, healing agents and chemotherapeutics.

The antimicrobial may be one or more selected from the group comprising: copper, silver, cobalt and zinc.

The antiseptic may be one or more selected from the group comprising: peroxides ( such as dibenzoyl peroxide, H₂O₂), iodine (such as iodophores, chiniophon), amines and amides (such as Tego acids, biquanide derivatives), ammonium salts (such as cetyltrimethyl amnonium cation), sulphadiazinates (such as Ag, Zn sulphofodiazinates), thiazine (such as methylene blue) and acridine dyes (such as ethacridinium cation).

The antibacterial may be one or more selected from the group comprising: chlorohexidine, iodine, chiniofon, gentamicin, bacitracin, neomycin and polymyxin B.

The healing agent may be one or more selected from the group comprising: hyaluronic acid, aloe vera, tissue engineering actives such as living cells and sacrificial materials such as collagen.

In one embodiment the additives may be impregnated into the mesh.

^■ Alternatively, the additives may form a biocompatible intermolecular complex (IMC) ■ with the oxidised polysaccharide. The biocompatible intermolecular polymer complex may be a complex of: an anionic component comprising polyanhydroglucuronic acid, which is a partially or completely hydrolysed in a normal and/or oxidative environment; and

a cationic component comprising one or more of the additives.

The nanofibres of the product as may contain a modifier and/or an emollient.

The modifier may be selected from the group of monomer or polymer acids, hydroxyacids, such as tartaric acid, citric acid, malic acid or malonic acid, succinic or maleic acid or their isomer fumaric acid or mixture thereof.

The emollient may be selected from the group of polyhydroxycompounds and/or aminoalcohols such as glycerol and polyglycerols, ethylene and propylene glycols and low molecular polymers and copolymers thereof (e.g. poloxamer 407) or aminoalcbhόls, e.g. mono-, di-, triethanolarnine and mixture thereof.-

At least one layer of the product may be a carrier layer. The carrier layer may be a textile.

The product may be biodegradable.

The product may be a wound dressing.

Definitions

The invention in particular involves polyanhydroglucuronic acids, salts and intermolecular complexes (IMC) thereof. The term polyanhydroglucuronic acid, salts and IMCs thereof as used herein also includes copolymers thereof, for example anhydroglucose copolymers. These as a whole are hereinafter referred to as PAGA. Patent applications numbers CS 242920, CS 292723, GB 2314840, and WO98/33822 (the entire contents of which are herein incorporated by reference) describe polyanhydroglucuronic acids and salts thereof and a method of preparing such compounds. The term polyanhydroglucuronic acids and salts thereof includes the acids and salts referred to in these patent applications.

Other proteins, peptides or aminoglycans of significantly higher molecular weight for example a molecular weight greater than 75kDa (such as collagen, chitin or chitosan) can also be used to prepare PAGA intermolecular complexes.

The partial or complete hydrolysis and neutralisation or ion exchange of the PAGA containing material is carried out in aqueous or water based organic solutions of inorganic or organic salts and bases and/or an oxidative environment. A stable PAGA product with a reduced degree of crystallinity and a high degree of purity in a microdispersed form is produced. WO 00/05269 describes intermolecular complexes, the entire contents of this document is herein incorporated by reference.

Brief Description of the Drawings

The invention will be more clearly understood from the following description of some embodiments thereof, given by way of example only with reference to the accompanying drawings in which:-

Figs. 1 A to C are schematic representations of a wound care dressings for surface applications. Fig. 1 D is a schematic representation of an absorbable dressing for internal applications (after removal of the supporting cover layer) Figs. 1 E to G are schematic representations of alternative wound care dressings; Fig. 2 illustrates the structural formula of oxidised cellulose. In the case where parameters y;z = 0 in the formula it is glucan, in this case cellulose. In case of parameters value x;z = 0 the formula represents PAGA (polyanhydroglucronic acid), and its salts or IMC (intermolecular complexes). The symbols Ma⁺ and Mb²⁺ or preferably Mb^x+ indicate the presence of mono and divalent (respectively polyvalent) cations;

Fig. 3 is an electron micrograph of the basic particles of PAGA Ca/Na salt having a size of 30-50nm agglomerated to spheres with having a size of 500-900nm;

Fig. 4 is a detailed view of granulation components in use. There are nanofibres with a large population of macrophages having different functional activity. (Enlargement x 200, alcian blue-hematoxylin-eosine);

Fig. 5 is a histological image of granulation tissue central area from experimental wound subcutis. There are numerous fibroblasts and fibrocytes and many sections of newly created capillaries (Enlargement x 200, alcian blu - hematoxylin-eosine);

Fig. 6 is a photograph illustrating the application of nanofabrics on liver tissue damaged by incision with accompanying strong bleeding;

Fig. 7 is a photograph of an open periotoneal cavity on the tenth day after the nanofabric application to the damaged liver tissue. The nanofabric is fully absorbed: the liver tissue defect is completely healed up. The peritoneal epithelium is free from any adhesions;

Fig. 8 is a photograph of a wound ten days after subcutaneous nanofabric application. The subcutaneous tissue and rectus abdominal muscle facies do not display any pathologic changes; Fig. 9 is an electron micrograph showing the size and shape of nanofibres prepared according to Example 1 (PVA - PAGA Ca/Na salt system);

Fig. 10 is a graph showing the tensile curve of nanofϊbre layer prepared according to Example 1 ;

Fig. 11 is an electron micrograph showing the size and shape of nanofibres prepared according to Example 8 (polyurethane - PAGA Ca/Na salt system);

Fig. 12 is a graph showing the tensile strength of a nanofibre layer according to Example 8 (polyurethane - PAGA Ca/Na salt);

Fig. 13 is an electron micrograph showing the size and shape of nanofibres prepared according to Examples 2 or 3 (PVA - PAGA Ca/Na salt system) after 24 hours exposure to saline solution. Crosslined nanofibres create pseudogel structures. In living organism they are readily absorbed; and

Fig. 14 is a graph showing the activation of the blood coagulation cascade by PVA/PAGA Ca/Na nanofibre and PAGA Ca/Na powder.

Detailed Description

The invention will be more clearly understood from the following description thereof given by way of example only.

The oxidised polysaccahride material is preferably in the form of biocompatible anionic polyanhydroglucuronic acid (PAGA), salt or IMC thereof. The PAGA may be prepared by partial or complete hydrolysis and neutralization in solutions of (in)organic hydroxides_., salts or bases in an aqueous and/or aqueous/organic system (CZ242920), or in an oxidative environment (GB2335921). During the preparation process salts are formed, for example salts with Na ions, or complex salts for example with Ca, Al, Fe ions but also with amino acids of lysine, arginine, or histidine types. IMCs are also formed for example with gelatin or peptides of hydrolysed collagen, but also with blood proteins or aminoglycans. The salts and IMCs may be formed by tuning hydrolysis conditions and types of salt, base and /or the mixtures used. .

The polysaccharide material may be polyanhydroglucuronic acid, biocompatible salts thereof, copolymers thereof or a biocompatible intermolecular complex polymer thereof. Preferably the oxidised polysaccharide is derived from cellulose, starch, or gum, or is of microbial origin.

PAGA displays a reducing ability in a biological environment similar to hyaluronic acid (HA). However PAGA has a higher assimilable organic carbon (AOC) value.

As such, PAGA and derivatives thereof prepared by the above methods are capable of forming a highly hydrated film on a biological surface such as the gastrointestinal tract (GIT) mucous tissue.

Hydrolytically prepared PAGA yields low viscous water solutions however, its IMCs with for example collagen, chitosan or other polymeric cations have a high viscosity. Such IMCs may be created as products during the hydrolysis process or afterwards in situ such as - on the mucous tissue of the gastrointestinal tract. IMCs may be formed from administering PAGA and the administered PAGA reacting with peptides or proteins present in ingested food. PAGA IMCs display a higher osmolality than simple PAGA salts (such as Na salt).

Nanofϊbres may be formed in the electrostatic field from aqueous or non-aqueous solutions and/or colloido-dispersed systems comprising fibre-forming polymers with PAGA and its derivatives. PAGA and its derivatives are dissolved in these solutions and/or create colloido-dispersed systems with a conductivity suitable for forming nanofϊbres. The nanofibres can be used to create separate fabrics or they can be used for covering of suitable base materials such as (nonabsorbable flat materials like (non)woven (non)laminated fabrics, films, foils or pads such as are used especially in wound dressing materials.

Production of different chemical substances in the form of nanoparticles or forming of suitable types of fibre-forming polymers into nanofibres is a new and very progressive way of preparation of materials with excellent properties. The particles of such materials have dimensions within units of tens or maximally hundreds of nanometres, and show different properties namely a greater specific surface area, higher porosity and a small pore size in comparison with the original materials. The properties may be as a result of, for example, a high specific surface area, fine crystalline structure, inner active surface size or porosity of the particles. New types of accelerators based on nanoparticle technology show higher specificity and higher effectiveness. This property creates a substantially higher efficiency of the given chemical reactions and their yields. Carbon in the form of nanoparticles is used as filling agent of composite materials. For instance, in medicine there is known usage of iron nanoparticles for monitoring of activity and diagnosis of cerebral tissue. Gold nanoparticles can be used for tumour tissue detection (source: University of California — San Francisco, Date 2005-06-06). Nanoparticles of cyanoacrylates may be used as excellent carriers of active substances - pharmaceutics.

Nanofibres are considered to be fibres with a diameter value lower than 1 μm in the non- woven textile industry. The NSF (National Science Foundation) Organization defines their smallest dimension (i.e. length or diameter) more specifically using a value below the level of 0,1 μm (=100nm). Nanofibres represent a new type of textile material that may be used in different branches of technology such as medicine, personal care, gas and liquid filtration or fuel elements. The high porosity value of some nanofibres types provides them with excellent heat-insulating properties. Some of the special properties of nanofibres predisposes them for use in medicine. For example, carbon nanofibres that have smaller diameter than erythrocytes can be used as a medicament carrier in blood cells [I]. The medical company STAR Inc. recently announced that they are able to deliver anti-adhesion materials in the form of nanofibres [2] Nanofibres for use in such procedures are prepared by an electrospinning process from a solution of a suitable biopolymer. In the case of cellulose-based polymers this process of formation might subsequently be affected by oxidation systems that transform a part of hydroxylic groups to aldehydic [7] or carboxylic [US 6,800,753] groups. The disadvantage of these processes is that oxidation takes place in a heterogeneous medium and thus it creates problematic end products whereby the fibres can also contain crystalline non-oxidized parts of cellulose fibrils. Cellulose and its common derivatives (HEC, HECMC₅ CMC etc.) are not degradable in the organism because the human organism does not contain cellulose enzymes.

Nanofibres may be prepared in two different ways:

1 a) Electrospinning process which makes use of electrostatic and mechanical force to spin fibres from the tip of a fine orifice or spinneret. The spinneret is maintained at positive or negative charge by a DC power supply. When the electrostatic repelling force overcomes the surface tension force of the polymer solution, the liquid spills out of the spinneret and forms an extremely fine continuous filament. It has the misleading appearance of forming multiple filaments from one spinneret nozzle, but current theory is that the filaments do not split. These filaments are collected onto a rotating or stationary collector with an electrode beneath of the opposite charge to that of the spinneret where they accumulate and bond together to form nanofibre fabric. Compared to electrospinning, nanofibres produced with this technique will have a very narrow diameter range but are coarser, as it is mentioned in the company publication of the company HILLS INC.

Ib) Electrospinning process and another technique for producing nanofibres is also the spinning of bi -component fibres such as Islands-In-The-Sea fibres in 1-3 denier filaments with from 240 to possibly as much as 1120 filaments surrounded by dissolvable polymer. Dissolved polymers leave the matrix of nanofibres, which can be further separated by stretching or mechanical agitation.

2) US 6,520,425 and US 6.382.526 disclose a method of nanofϊbre production from a polymeric solution using air flow. The hypothesis is that the nanofibre is created using a double capillary when the nanofibre is formed by a flow of gas supplied to the inner capillary. These production methods are characterized by a low output and a high process failure rate upon increase of capillary jets amount.

Patent applications US 2002/0175449, US 2002/084178, US 2004/0234571, US 2005/0095695 and US 2006/0046590 disclose a method of creating nanofibres using electrostatic field with an average intensity of 50-500kV/m. However the processes provide a very low output, i.e. a very low amount of processed polymeric solution.

WO 2005/024101 (the entire contents of which are incorporated herein by reference) describes a method of nanofibre production in the electrostatic field that noticeably eliminates disadvantages of other processes and increases the nanofibre production output.

Nanofibres can be prepared from different types of polymers in two different ways: a) nanofibres applied from aqueous medium b) nanofibres formed from polymeric solutions in organic solvents

Biodegradable polymers or other biodegradable materials known to the art may be used as a biodegradable matrix. Some examples of suitable biodegradable polymers are alpha- polyhydroxy acids, polyglycolide (PGA). poly(L-lactide), poly(DX-lactide), poly(.epsilon.-caprolactone), poly(trimethylene carbonate), poly(ethylene oxide) (PEO), poly(.beta.-hydroxybutyrate) (PHB). poly(.beta.-hydroxyvalerate) (PHVA), poly(p- dioxanone) (PDS), poly(ortho esters), tyrosine-derived polycarbonates, polypeptides and copolymers of the above-mentioned ones. A wide range of polymers, including biodegradable, polyesters, polyethylene, polypropylene polyamides and nylon. (Fibre system SMS). Previous to this patent application, all known biocompatible, biodegradable and bioresorbable polymers have been produced by jet nanofibre systems.

However, expensive equipment and patient discomfort are disadvantages of creating nanofibres directly on the body. In addition, not all the mentioned polymers are fibre- forming or at least film-forming and the solutions for the spinning process can not always be prepared in cheap and environmentally friendly solvents. As a result it is necessary to modify the working solution of the biopolymer by the addition of different additives prior to the spinning process. It is also necessary to use expensive and environment-unfriendly toxic solvents (hexafluoroIPA for dissolving poly(L-glycolide)copolymers, ,or N- morpholine, N-oxide, CS₂, cadoxen or cuoxam for cellulose solutions spinning).

Now we have found that nanofabrics, applicable especially in pharmacy and medicine, can be prepared from solutions of common cheap polymers that are biocompatible and nontoxic for organisms and that can be (but do not need to be) absorbable themselves in the live organism. Such nanofibres can be prepared from systems that are created from solutions of fibre-forming polymers. Oxidized polysacchande such as glucan that is soluble in the system or is in the form of nanoparticles that create colloido-dispersed systems of required viscosity and conductibility suitable for preparation of nanofibres using electrostatic spinning (Electrospinning method) may be included in the nanofibre.

The method of preparation of products that are partially or fully absorbable in live organisms and the products themselves advantageously use the combination of fibre- forming biocompatible (although originally nonabsorbable or which can only be partially eliminated from the organism) polymers and oxidized glucans. The nanofabric can be absorbed simply without any adverse side effects.

In this invention we describe nanoiϊbrilar structures of oxidized cellulose. These products especially in the form of nanofibres can be used as hydrating, haemostatic or anti-adhesive layers applied to commonly available dressing materials such as bandages for covering of minor injuries. They can also be used as the basis for new dressing materials suitable for both acute and chronic wound management. Such dressings may incorporate multifunctional capabilities by virtue of different types of nanofabric layers which can be combined with other fabric layers. For example, they can be used for dressing of chronic wounds such as covering of venous ulcerations and other wounds that are difficult to heal. In this case the undercoating bandage layers can be selected on the basis of which nanofibres are to be applied, for example, nanofibres can be selected from the group of biocompatible supersorptive fabrics on the basis of acrylates or an absorbing nonwoven pad based on polyether sulphone (PES) or regenerated cellulose or other cellulose derivatives as well as gelatine and polyurethane (PUR) foams. These materials can be used for creation of "sandwich-type" dressing materials with a haemostatic function, high value of early exudate sorption. The dressings can have a protective layer of non-woven water-insoluble nanofabric on the upper bandage part that enables gaseous components such as CO₂ and O₂ to diffuse through the bandage whilst preventing wound contamination by viruses and microorganisms.

Possible dressing constructions are illustrated schematically in the Fig. 1.

Fig. 1 - Examples of Dressing Construction

Fig. 1 represents various combinations of discrete layers including the following: covering layer (1) - an insoluble nanofibre mesh, e.g. polyurethane. polyacrylic absorbing layer (2) - a superabsorbing fabric (not necessarily nanofibre). e.g. acrylic, polyester gelforming layer (3) — a crosslinked nanofibre mesh haemostatic layer (4) - a non-crossJinked nanofibre mesh. e.g. PVA + M.D0C^1M Na salt (M.DOC™ is supplied by Alltracel Pharma Ltd. Dublin) antibacterial layer (5) - a nanofibre mesh involving antiseptics such as chlorohexidine, iodine, chiniofon, gentamicin

Figs 1 (a - c) illustrate wound care dressings for surface applications

Fig. l(d) illustrates absorbable dressings for internal applications (after removal of supporting cover layer (6))

Due to the low mass of nanofibre (typically 1 to 30g/m² ) and the very easy application over large areas, production of the dressings of Fig. 1 is very cost effective and does not add significantly to overall product cost. This is a major advantage of the invention over previous technologies used to incorporate oxidised glucan and associated derivatives into woundcare products. Such nanofabrics can be applied to the surface of any type of dressing material and may be (but not necessarily) fully absorbable. Any biocompatible polymer can be used as a carrying polymer in the system.

Nanofabrics of the invention can be used in a wide variety of applications. They can be applied, for example, like tectorial membranes in stomatology for covering- of damaged soft tissues. In connection with chlorhexidine as a known antiseptic agent they may be used for the reduction of halitosis, or the reduction of damaged bleeding gums. The nanofϊbres may be in the form of "rolls" or "cut fibres" and can be used as a hemostatically effective pin which can be placed into a hole after tooth extraction. The product is also suitable for prevention and treatment of paradontitis.

In one case the nanofibres are applied on a suitable dressing material, which may be used for covering of chronic wounds. The nanofibres themselves may be prepared according to the invention on a handling pad. The handling pad may be removed after the application of nanofibres (for example, a polypropylene spundbond). Such products may have a wide range of uses in surgery, for example for covering surgically damaged internal organs in body cavities. The products stop heavy bleeding (for example, upon liver tissue damage). The products can also serve as anti-adhesives. In contrast to other known anti- adhesive materials such as Interseed TC7 or TC7Na (company Johnson & Johnson), FloGel of the company Aliance Pharmaceutical Corp. or Seprafilm (Enzyme Corp) the products of the inventionshow high effectiveness.

According to the invention other materials such as antimicrobial substances, antibiotics or other chemotherapeutics can be added to nanofibres directly or in various layers. The substances can be added as a filling material that can be released from the product by simple diffusion through a polymeric membrane. Alternatively, they can create IMCs with oxidized glucan present in the nanofibres. In addition to diffusion processes the release of nanofibres int the wound and effectiveness of the dressing is dependant on the speed of biodegradation of the oxidized glucan. Antiseptic agents such as peroxides (dibenzoyl peroxide, H₂O₂), I₂ (iodophores, chiniophon), amines and amides (Tego acids, biquanide derivatives), ammonium salts (cetyltrimethyl amnonium cation), sulphadiazinates (Ag, Zn sulphofodiazinates) or thiazine (methylene blue) and acridine dyes (ethacridinium cation) are examples of common chemotherapeutics that may be used. Antibiotics may be incorporated into the dressing (nanofibre) for local application. For example polypeptide preparations of antibiotics (for example, bacitracin Zn, polymyxin B) or aminoglykosides antibiotics (for example, neomycin, gentamycin).

Surprisingly oxidized glucan salts with Co, Zn, Ag or Cu ions show antiseptic properties at concentrations that are not toxic or irritating to tissue. For example, at concentrations within the range of 0. 1% - 3% of the oxidized glucan mass, the oxidised glucan salts have an bacteriostatic and fungistatic effect.

The nanofabrics may be used in a single layer or may comprise multilayers (sandwich) to provide a product simulating cell growth in vitro (for example, growth of chondrocytes, keratinocytes. fibroblasts etc.). for covering of surgical wounds, and/or as anti-adhesive preparation used for prevention of post-surgical adhesions in body cavities. The nanofabrics in accordance with the invention are prepared from solutions of fibre- forming polymers and oxidized polysaccharide glucans such as oxidized cellulose or its derivatives are the preferred oxidized glucans.

Oxidized cellulose is a static ionogenous copolymer of glucose and glucuronic acid (Fig. 2).

GB 2 335 921 discloses a method of preparing a high quality microdispersed oxidized cellulose.

Fig.2: Oxidized cellulose formula

In case of parameters value y;z =0 the formula represents a glucan, in this case cellulose. In case of parameters value x;z =0 the formula represents PAGA (polyanhydroglucuronic acid in the form of β-D anomers), eventually its salts or IMC (intermolecular complexes). The symbols Ma⁺ and Mb²⁺ or preferably Mb^x+ represent the presence of mono and divalent (respectively polyvalent) cations.

The conductivity of the working solution can be optimised using a selection of suitable types of PAGA salts or IMC without necessarily using other foreign auxiliary substances, acceptance of which would be potentially questionable from the point of view of pharmacology, as such the use of inorganic salts is a considerable advantage of the present method of nanofϊbre preparation.

If it is required, the groups -C=OH can be introduced to the molecule, for example by using HIO₄ or its salts such as NaIO4 or KIO4 instead of H₂O₂ as an oxidative agent during hydrolysis according to the method described in GB 2 335 921.

Nanofibres are created from pre-defined polymers, i.e. from carrying fibre-forming polymer, and defined PAGA derivative is another important advantage of nanofibres according to the invention. Using this approach required nanofibre physical and biological properties can be preset. The PAGA particles define pores with a large specific surface area in insoluble systems. This enables penetration of exudate with enzymes to the nanofibre and consequent relatively easy biodegradation. During extensive testing of PAGA properties prepared according to the method of GB 2 335 921 and its derivatives we have established the following:

• PAGA, its (in)organic salts, complex salts or IMC can be prepared from arbitrary glucan with established uronic carboxylic group in the C₆ position of the anhydroglucose unit of the chain using an arbitrary type of oxidation. Such uronic derivative is processed as raw material using a procedure according to the method of GB 2 335 921 into nanoparticles or microparticles having a size from 30 nanometres to 5000μm according to given requirements (particle size up to lOOOnm is preferred for nanofibre production). The particles show a specific surface size of up to 200m²/g. During the production they can be agglomerated to spheres (see Figure 3). Unlike oxidized glucan the crystallinity level of which ranges usually within 20-60%, PAGA and its derivatives are fully amorphous polymers. This enables controlled biodegradation of nanofibres in a live organism. The suitable size of PAGA particles if PAGA is applied in non-aqueous spinning systems is generally within the range of 3O-5OOnm.

• PAGA and its derivatives prepared in this way are very hydrophylic polymers that create solutions and colloido-dispersed systems in an aqueous environment (this depends on the level of polymerisation, type of salt or IMC). Probably due to. the impact of hydrophobic interactions it is mixed very well with non-polar substances such as oils, greases or hydrophobic polymers. This phenomenon can be exploited in systems of nanofibres production according to the invention. For example, for preparation of colloido-dispersed non-aqueous systems for nanofibre spinning in the electrostatic field using the procedure disclosed in WO 2005/024101. Fig. 3 illustrates basic particles of PAGA Ca/Na salt (M DOC™) with the size of 30- 50nm agglomerated to spheres with the size of 500-900nm.

• On the basis of extensive biological testing we have found that PAGA and especially its Na, Ca salts and IMC with, for example, gelatine peptides increase non-specific immune response of the organism. PAGA Ca/Na salt under in vitro cultivation was found to have a substantial effect on spontaneous proliferation of mouse splenocytes isolated from both inbred strains of mice. This effect depends on concentration [5]. PAGA and its biocompatible derivatives strongly support accumulation of macrophages (Mf) and their proliferation in live tissue where they are applied. They serve as a substrate for Mf. Surprisingly the nanofibres prepared from systems according to the invention have the same effect, i.e. the nanofibre is created by carrying originally non-absorbable or upon certain conditions partially absorbable fibre-forming polymer with PAGA and/or its derivatives. This phenomenon may cause full absorbability of nanofibres prepared according to the invention. (See Figure 4)

Fig. 4 is a detailed view of granulation components in use. There are nanofibres with a large macrophage population with different function activity. (Enlargement x 200, alcian blue-hematox3'lin-eosine)

• PAGA and its derivatives as well as nanofibres prepared from them are highly angiogenetic, thus they increase blood supply of tissues, which results in acceleration and improvements of all stages in the healing process. (See Figure 5)

Fig. 5 is a histological image of granulation tissue centra] area from experimental wound subcutis. There are numerous fibroblasts and fibrocytes and many sections of newly created capillaries (Enlargement x 200. alcian blue-hematoxylin-eosine)

• PAGA and its derivatives create hypoosmotic systems in aqueous solutions. • In comparison with, for example, HA the aqueous solutions of PAGA and its derivatives show approximately 1Ox lower viscosity upon quite high molecular mass upon the same concentration (According to GPC determination within the range of approximately 5.10³ to 5.10⁵ daltons), in solutions with the same concentration.

• PAGA can create IMC with a wide range of counter ions, especially from the group of medicaments (antimicrobial substances, antibiotics, anaesthetic agents, cytostatic agents etc.)

• PAGA and its derivatives are fully absorbable without any side-effects. When used on a wound they can have a haemostatic effect, especially in case when at least one cation bound to PAGA is a multivalent ion, especially Ca, Al, Fe. Such derivatives accelerate especially the primary haemostatic process, i.e blood platelet aggregation. In case of IMC the PAGA with some peptides has an anticoagulation effect [6]. The same effect is shown by nanofibres produced from these materials. (See Figures 6 to 8)

Fig. 6: Application of nanofabiϊcs on liver tissue damaged by incision with accompanying strong bleeding

Fig. 7: View of open peritoneal cavity on the tenth day after the nanofabric application to the damaged liver tissue. The nanofabric is fully absorbed; the liver tissue defect is fully healed up. The peritonea] epithelium is without any adhesions

Fig. 8: Experimental wound after ten days of subcutaneous nanofabric application. The subcutaneous tissue and rectus abdominal muscle facies are without any pathologic changes

» The PAGA contains reactive groups (especially secondary OFI and/or primary and secondary COOFI groups) that can mutually react upon a suitable accelerator. These PAGA chain groups can also react with the same or other groups such as - NH₂, -CO NH₂ or -COOR that are the part of the polymeric chain for the spinning system of the added carrying fibre-forming polymer. These polymeranalogous reactions, especially of a condensation type, enable alteration of the biological, physical and chemical properties of the nanofibre.

• PAGA and its derivatives in the form of nanoparticles create colloido-dispersed solutions even in organic solutions, especially those that are at least partially mixable with water. Suitable organic solutions may be selected from the group comprising: dihydroxy compounds such as polyolefine oxides (polyethylene oxide, polypropylene oxide and their copolymers, their ethers like ethylene glycol (mono) dimethyl ether and the like, (poly) alcohols such as C_1-4 alcohols, ketones, especially acetone, glycerine and polyglycerols, amides and substituted amides like formamide, dimethylformamide, acetamide, dimetylacetamide, tertiary amines such as triethanolamine, pyridine, N-methylpyridine, N-methylpyrolidone.

More suitable organic solutions that may be used can be derivatives of esters such as methyl, ethyl, butyl or amyl acetates and/or their mixtures, of from acids such as HCOOH, CH₃COOH.

• Water and/or systems of the above-mentioned solvents are the generally appropiate solvents for PAGA and their derivatives.

• Nanofibres formed using an electrospining method, especially according to the procedure disclosed in WO 2005/024101, can be produced from different types of fibre-forming polymers. For the purposes of nanofibres production according to the invention they can be divided into two groups: a) systems soluble in water b) systems soluble in organic solvents.

The first group consists of the following substances: derivatives of alginic acid such as glycolalginate or sodium alginate, pectines_.. high-molecular dextran, pullulan or chitosan, vegetable gums or gels of xylan or glucomannan types etc., from the group of proteins and peptides, especially collagen and gelatine (bovine but also fish gelatine), from synthetic water-soluble polymers the selection is poly(meta)acrylic acid and its salts, poly(met)acrylamide, polyhydiOxypropyl(met)acrylamide, polyvinylpyrrolidome, polyethylenimines, polyallylamine, then copolymers of maleic or fumaric acid with styrene or their terpolymers with acrylic acid or its derivatives in the salt forms.

From the group of polyhydroxy mixtures synthetic water soluble chemical entities such as: polyvinylalcohol; high-molecular poly(ethylenglycols) with a value of molecular weight higher then 3.10⁴ Daltons; water-soluble derivatives of cellulose such as carboxymethyl, carboxymethylhydroxypropyl, methyl, hydroxypropyl cellulose; natural silk ; proteins; or mixtures of these polymers can be used in the electric spinning system.

The second group contains the following substances: cellulose and their water- > soluble derivatives such as triacetate, acetobiityrate or formiate, chitin, polylactic ' acid and its copolyesters with glycolic acid, polyamides like 6,6, maleic anhydride copolymers with styrene or their terpolmers with acrylic acid and its esters, fibre- forming polyurethanes, poly(met)acrylic acids.

The ability of this second group of substances to create biocompatible nanofϊbres using electrostatic spinning is the only limiting factor of spinning systems with PAGA content according to the invention. Such systems should have corresponding viscosity, dry weight, mol. weight and suitable conformation arrangement of the carrying fibre-forming polymer. The spinning system dry weight usually ranges between 2%-25%. viscosity (η) within the range of lOOmPas - 1000 mPas. electric conductivity within the range of 10-50 mS/cm. spinning solution surface tension within 20mN/m-50 mN/m. These values range with advantage within the ranges of dry weight 5%-15%. η 200mPa.s-300 mPa.s. conductivity 20mS/cm-35 mS/cm and surface tension value in the range of 30mN/m-40 niN/m.

The list of fibre-forming polymers suitable for nanofibres produced according to the invention that are mentioned in two groups outlined above is by way of example only, because the preparation of biocompatible nanofibres using electrostatic spinning from spinning systems created by any suitable .type of spinning polymer in a suitable solvent that contains PAGA or its derivative in the form of nanoparticles or in the form of their solution in the spinning system is within the scope of the present invention.

In order to modify properties of prepared nanofibres the spinning system can contain other components as spinning accelerators, softeners or other substances that may participate in the spinning reaction. Biological compatibility is a condition for application of these substances in the spinning system.

PAGA can easily create lactones between the carboxylic group and hydroxylic group on the carbon C₃ of the anhydroglucuronic unit - C_OOOC₃ — of the same chain as well as the ester bond by the reaction of C₆OOH carboxyl with the hydroxylic group on C₃' of the anhydroglucuronic unit of the adjacent chain. Spinning can be used, for example, upon preparation of partially soluble PAGA gels. In order to achieve increased nanofibre strength, the condensation reaction between PAGA and reactive groups, especially the aminie, amidic. carboxylic or hydroxylic ones contained in the chain of carrying fibre-forming polymers is more suitable. The following substances belong to the group of reactive polymers with the ability to create nanofibres according to the invention: polyvinyl alcohol, polyoxyeters (polyethylene oxide, polypropylene oxide, their copolymers. polyhydroxypropyl(met)acrylamide.. polyhydroxyetyl(met)acrylate, hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropylcarboxymethyl cellulose, carboxymethyl cellulose, poly(met)acrylamide, polyurethanes with final hydroxylic or carboxylic groups, pectines, alginic acid or their blends .

Although known substances like biscarbodiϊmides US 6,096,727, dihydrazides WO 03/006068, divinylsulphone US 4,582,865, or tetraalkylamonium salt of PAGA can be used for nanofibre spinning according to the invention the condensation reactions of PAGA - PAGA and PAGA - carrying fibre-forming polymer upon creation of ester or amide bonds accelerated by Lewis acids are preferred.

Condensational substances such as monomelic acids and hydroxyacids such as 1,2,3,4-butanetetracarboxylic acid, tartaric acid, citric acid, malic or malonic acid, _. succinic and maleic acid or their derivatives and mixtures can be used for modification of application system properties as well as for modification of biological and physical-chemical properties of nanofibres with PAGA content according to the invention, of bϊocompatibilily the catalysis especially Of H₃PO₄ ^:< or H₂SO₄ is preferred. Both are within the scope of the invention.

Also (in)organic cations present in the solution of polymers , or colloido-dispersed system with PAGA content according to the invention that, impact on values of conductibility and viscosity and in this way on diameters of -prepared nanofibres as well as on their biological properties, especially on anticoagulation or haemostyptic effects or speed of absorption from the organism can be used for the same purposes. For example, the presence of polyvalent cations such as Ca, Ai, Fe increases haemostatic effectiveness of prepared nanofibres. . The presence of basic amino acids like arginine, lysine, histidine or glutamine as well as of organic amines reduces this haemostatic effectiveness.

Especially polyhydroxy mixtures such as glycerine and polyglycerols. ethylene and propylene glycol and their low-molecular polymers and copolymers (for example, poloxamer 407) or amino alcohols such as mono, di, triethanolamine can be applied as softeners and modification additives in nanofibres according to the invention. If the prepared nanofibre undergoes the netting reaction these substances can participate in this netting reaction and can also serve as modifiers of physical mechanical properties.

Physical properties of nanofibres according to the invention can be modified considerably. This is illustrated by two example formulations: Example 1 (Figures 9&10 and Table No. 1) has considerably less fibre elongation as compared to the far greater elasticity shown in Example 8 (Figures 11&12 and Table No 2) Both nanofibre samples were tested across a width of 25mm and have a basic weight of 5g.nϊ².

Fig. 9: Size and shape of nanofibres prepared according to Example 1 (PVA / PAGA Ca/Na salt system)

Tablel : Strength and elongation of nanofibrous samples in cross direction prepared according to Example 1

Fig. 10: Tensile curve of nanofibre layer prepared according to Example 1 Fig. 11: Size and shape of nanofibres prepared according to Example 8 (polyuretliane / PAGA Ca/Na salt system)

Table 2: Tensile strength and elongation of nanofibroυs samples prepared according to Example 8 in cross direction

Fig 12: Tensile curve of nanofibre layer prepared according to Example 8 (polyurethane - PAGA Ca/Na salt)

Fig 13: Size and shape of nanofibres prepared according to Examples 2 or 3 (PVA - PAGA Ca/Na salt system)

The invention will be more clearly understood with reference to the following examples.

M.DOC -.TM Nanofibers - formulation examples Examples

Meshes comprising nanofibers of oxidised polysaccharide were prepared by electro spinning using the method described in WO 2005/024101. These sample formulations were prepared as prototypes to demonstrate the range and capability of nanofibre based materials in a range of possible medical devices.

Example No. 1 Haemostatic wound healing manofibre ϊayer (weakly cross-linked) for conveBtioiaaϊ woirad pads used in primary care.

Raw material:

SIovMR 16% water solution of PVA (supplier CHZNovaky, Slovakia)

Grade of hydrolysis 88%

Saponification 141mgKOH/g

Specific conductivity 310μS/cm

Dry matter 15,9%w/w

M_w . 85 IcDa

Potyanhydroglucuronic acid Ca²^Na⁺ salt PAGA (supplier Alltr&cel Phurma Ltd.

Dublin)

Loss on drying 2,6%

Carboxyl Content 20_;6%w/w

Ca²⁺ Content 5,6% w/w

Na^"* Content 2,7%w/w

Water solubility 100% for concentration 15%w/w pH 1 %( w/w) water solution 5,8 M_w 5IkDa

Particle size 100-500nm

PVA /PAGA Ca/Na ratio (proportion): PVA 60 parts

PAGA Ca/Na salt 40 parts

Catalyser none

Procedure:

16Og of powder PAGA salt is added into the mixing container of fibre-making equipment (as described in WO 05/024101) and mixing starts. 840ml of demineralised water is added. When PAGA salt is completely dissolved, 150Og of PVA (Sloviol Rl 6) water solution is added. Viscosity is adjusted by addition of demineralised water to a target of 250mPa.s at 20°C. The conductivity is adjusted to 24mS/cm, surface tension for 41mN/m. The polymer solution then flows into a trough container with 1000mm length, where the cylinder with rifts is used as an applying electrode. The cylinder rotates at a speed of 3 rpm. Constant layer of the solution in trough container is checked by ultrasound sensor, circulating pump and three way valves. Cylinder electrode is connected to D. C: with voltage of 4OkV,. Counter electrode is wire net.., that follow the vacuum chest. There is a belt of a carrier, permeable textile, that is fastened at the counter electrode under pressure made by vacuum chest (suction, box). The rotating cylinder electrode brings up the fibre-making solution from the trough container and by electric field so-called "Tailer cones^"' are made. By this method nanofibres with diameter 400- 500nm are made.

The nanofibres are applied to a carrier textile (PES-polyester non-woven textile 300g/m²). The layer of nanofibres is dried by air flow at' 6O⁰C that is between, both electrodes. Speed of production is about 2g/min. ^' • ,

The carrier textile with the layer of nanofibers having a weight of 5g/m² can be used for the preparation of wound dressings with haemostatic and wound healing effects. This non crosslinked nanofibre layer provides rapid delivery of a haemostatic gel layer (when contacted with wound exudate), and ensures wound healing in the initial moisture phase. Example No. 2 Wound healing nanofibre layer as an alternate wound pad surface for longer term applications.

Raw material:

SloviolR 16% water solution of PVA (supplier CHZNovaky, Slovakia)

Grade of hydrolysis 88%

Saponification 141mgKOH/g

Specific conductivity 310μS/cm

Dry matter 15,9%w/w

Polyanhydroglucuronic acid Ca²^Na⁺ salt PAGA (supplier Alltracel Pharma Ltd. Dublin)

Loss on^' drying ^'■. ^' 2,6%

Carboxyl Content 20,6%w/w

Ca²⁺ Content 5,6% w/w

Na⁺ Content 2,7%w/w

Water solubility 100% for concentration 15%w/w pH l%(w/w) water solution 5,8

Particle size . 100-500nm

PVA /PAGA CaJNa ratio (proportion):

PVA 59 parts

PAGA Ca/Na salt 40 parts

Catalyses- 1 part (85% H₃PO₄ (Fluka index No. 04107)) Procedure:

The procedure is the same as in Example No. 1. When the polymer substances are mixed, catalyser is added.

The network of nanofibres is stable due to higher degree of crosslinking, so the dressing/ bandage can be left on the wound for 48 hours.

Example No. 3 Composite material of low crossMnked (weak) haemostat layer backed by stronger wound healing layer on a convential wound pad backing

Raw material:

- same as Example No. 1 and 2

Procedure:

Procedure is the same as in Example No. 1. and 2. As a carrying textile we used polypropylene' non-rwoven textile with a weight of 20g/m² (spunbond). Nanofibers are applied in two layers:

1. layer with weight 5g/m² nanofibers made by system in Example No. 1 (see above)

2. layer with weight 10g/m² nanofibers made by system in Example No. 2 (see above)

When the nanofibres layers are made, nanofibers are separated from carrying the textile and wound up into a roll. Cross linking can be improved by energy addition - heating (such as by sterilisation in autoclave as per EN285:1996) or by γ radiation with dosage 25kGy (as per EN 552:1994/A1 :1999; and A2:2000 "Sterilization of Medical devices - Validation and Routine Control of Sterilization by Irradiation^"), or by microwave or infrared radiation. This material can be used as a medical device to stop bleeding during surgery, for example, in the peritonea or the thoracic cavity. The material can be formed into a pack (such as by husting) and may be used to stop bleeding and wound healing after tooth extraction in stomatological or maxillofacial surgery.

Example No. 4 Composite nanofibre wound dressing incorporating Antiseptic

Multilayer material of nanofibres is made by a modified procedure as per Example No. 1.

The incorporation of the PAGA technology into the nanofibre form by electrospinning also allows the combination of other actives by incorporation into the wound dressing. Antimicrobials such as silver, copper, cobalt, chlorohexidine, etc may be added into the pad by intermolecular complexes with PAGA prior to electrospinning, by impregnating the pad or by addition of a nanolayer of the active. Such a matrix can kill clinically relevant bacteria in the dressing to help maintain bacterial balance and reduce bacterial growth, which may reduce the risk of infection. The dressing can also promote healthy tissue growth while delivering the antimicrobial to the wound. Such a combination multilayered product could only be produced with PAGA in the nanofibre form.

Other biological actives that promote healing such as aloe vera, tissue engineering actives such as living cells and sacrificial materials such as collagen can all be incorporated into the PAGA containing nanofibre dressing. This possibility greater expands the scope of the PAGA technology and brings the PAGA properties to these areas of healthcare. The potential to multilayer many actives into the dressing allows a delivery of a multitude of properties. An example of the building of such a technology can be seen in Figure J E to G in which: ^• layer 4 is a haemostatic layer comprising, for example, PAGA; layer 6 is a backing material comprising, for example, polyester; layer 7 is an antimicrobial layer comprising, for example metal ions such as silver, copper, cobalt, zinc and the like and combinations thereof; layer 8 is a healing layer comprising a healing active.

The multilayer dressing may have one or more layers of each type of layer for example the dressing of Fig IG has two antimicrobial layers 7 A and 7B. Layers 7A and 7B may be the same or different. For example, layer 7A may comprise one or more metal ions such as silver and layer 7B may comprise the same or different one or more metal ions such as copper. The multilayer products shown in Fig. 1 are for illustrative purposes and it will be appreciated that the combination and number of layers may be varied in accordance with the invention.

Nanofibres may be applied on polypropylene spunbond as follow: First layer — no cross linking weight 5g/ m²

Second layer - cross linking weight 15g/ m²

Raw material:

First, layer of nanofibres

Elvanof 52-22 PVA (supplier DuPont) -

Grade of hydrolysis 88%

Saponification 138mgKOH/g

Specific conductivity 305μS/cm

Dry matter 15,9%w/w

M_w 85 IcDa

Potyanhydroglucuronic acid Ca²^Na⁺ salt PAGA (supplier Alltracel Pkarina Ltd. Dublin)

Loss on drying 3,4% Carboxyl Content 23,3%w/w

Ca²⁺ Content 5,8% w/w

Na⁺ Content 3,0%w/w

Water solubility 100% for concentration 15%w/w pH l%(w/w) water solution 6,1

Mw 55kDa

Particle size . 100-500nm

PVA /PAGA Ca/Na ratio (proportion):

PVA 60 parts

PAGA Ca/Na salt 40 parts

Catalyser none

Second layer of nanofibres

Polyanhydroglucuronic acid Ca²^Na⁺ salt PAGA (supplier Alltracel Phartna Ltd. Dublin)

Loss on drying 2,7%

Carboxyl Content 14,8%w/w

Ag⁺ Content 26,2% w/w

Na⁺ Content 0₅7%w/w

Water solubility 100% for concentration 15%w/w pH l%(w/w) water solution 5,5

M_w 35kDa

Particle size 100-5 OOnm

PVA /PAGA Ca/Na /PAGA Ag/Na ratio (proportion):

PVA 60 parts

PAGA Ca/Na salt 39 parts

PAGA Ag/Na salt 1 part Catalyser none

Both nanofibre layers are applied on a viscose non-woven textile with a weight of 30Og/ m² with a covering layer (from the opposite side) of 5g/m² PUR nanofibers (see Figure Ia). This covering layer of PUR nanofibres prevents microbial contamination of the wound.

Procedure:

200ml of PAGA Ag/Na solution (5% in water) is made in a laboratory glass beaker.

1500ml of 16% solution of PVA Elvanol^® 52-22 is made in another glass beaker.

840ml of demineralised water is added into the container of nanofibers machine and slowly 16Og PAGA Ca/Na salt is added. When completely dissolved, 150Og of PVA water solution is added. When the^' solution is homogenous a solution of PAGA Ag/Na is added. Other steps are the same as per the procedure in Example No. 1 above.

A second layer of nanofibers is made using a formulation similar to the first layer. In this case we use as a cross linking catalyst 1 part of 85% H₃PO₄ . By slowing the speed we increase the weight of nanofibers to 1 Og/ m²'

This bandage material is then exposed to microwave radiation, this resulted in crosslinking of mainly the second layer of nanofibres. Both nanofibre layers have effective antiseptic properties against gram-negative, gram-positive micro-organisms, ferments and fungi. They also have high absorbing ability for exudate (absorption of about 2Og of exudate by 1 g of nanofibers). The bandage can be used for covering of chronic wound (mainly infected varicose ulcer).

Example No. 5 Flexible crosslinked nanofibre textile To improve crosslinking we used tartaric acid and for improved flexibility we used a mixture of glycerine and polyethylene glycol (macrogolum 1500).

Incorporation of polymers such as polyethylene glycol (PEG), polyethylene oxide (PEO) and/or PUR (polyurethane) can be added to the PAGA nanofibre to give the structure a physical conformability and flexibility. This will allow delivery of the PAGA to any relevant wound site through by the manipulation of the conformable dressing. This was not possible with the PAGA technology in the micronised form. Il will be appreciated that polymers such as PEG, PEO and PUR may be incorporated into any of the dressings exemplified herein.

Raw material: - see Example No. 1

PVA /PAGA Ca/Na/ other substances ratio (proportion):

PVA 45,8 parts (286d Sloviol R 16)

PAGA Ca/Na salt 31 parts

Tartaric acid 3 parts (FLUKA index No.95318)

Glycerine 11 ,65 parts (FLUKA index No.49779)

Macrogolum 1500 7,78 parts (FLUKA index No. 81214)

Catalyser 85% H₃PO₄ 0,77 parts (FLUKA index No. 04107)

Procedure:

PAGA Ca/Na salt and water is added into the container of the nanofibre machine. Mixer is switch on. When PAGA salt is completely diluted, the tartaric acid, glycerine, macrogolum and H₃PO₄ are added. When the solution is homogenous, PVA solution is added. Other steps are the same as in Example No. 1.

Example No. 6 Nanofibres with intermolecular complex of antibiotic - PAGA Ca/Na Raw material: - see Example No. 1

PVA /PAGA Ca/Na /other substances ratio (proportion):

PVA 49,62 parts (286d Sloviol R 16) .

PAGA Ca/Na salt 33,58 parts (see Example No. 1)

Glycerine . 12,63 parts (FLUKA index No.49779)

98%H₂SO₄ 0,92 parts (FLUKA index No. 30743)

Gentamicine sulfate 3,25 parts (FLUKA. index No. 48760)

Procedure:

PAGA Ca/Na salt and water is added into the container of the nanofibers forming machine and the mixer is switch on. When PAGA salt is completely dissolved, the gentamicine sulphate is added. When diluted, the intermolecular complex is made (higher viscosity), glycerine and PVA solution is added. At the end sulphuric acid is slowly added. Other steps are the same as in Example No. 1. The nanofibers are applied on the super sorbent textile type 2323 ("Technical Absorbents" company, UK).

The material can be use for wounds with heavy exudates and microbial contamination.

Example No. 7 Wound healing nanofibre layer as an alternate wound pad surface for longer term applications

Raw material:

PVA see Example No.2 (Sloviol Rl 6)

PAGA Ca/Na salt see Example No.2 (M.DOC™)

Polyvinylpyrrolidone K60 (45% in water-FLUKA_r index No.81430) PVA /PAGA Ca/Na/ other substances ratio (proportion):

PVA 45 parts

PVP 14 parts

PAGA Ca/Na salt 40 parts

Catalyser 85% H₃PO₄ 1 part (Fluka index No. 04107)

Procedure:

The procedure is similar to Example No. 1. When the polymer substances are mixed, the viscosity and conductivity are adjusted for required limits (22mS) and the dosage of catalyser is added. Cross linking reaction is proceeding in the roll during sterilisation process and storage for about 30 days at 20-25°C. The cross linking layer of nanofibres is stable, so the bandage can be on the wound for 48 hours.

Example No. 8 Alternate highly flexible haemostatic and wound healing nanofibre layer intended for use where high elongation is required (siscfe as stretch fabrics)

Raw material:

Polyurethane Desmoderm 4319 Bayer Material Science AG, 51368 Leverkusen N.N-Dimethylformamide Fluka index No.33120

Polyanhydroglucuronic acid Ca²⁺ salt PAGA (supplier Alltracel Pharrna Ltd. Dublin)

Loss on drying 1 ,9%w/w

Carboxyl Content 22,7%w/w

Ca²⁺ Content 8,8% w/w

Na⁺ Content 0,l%w/w water solubility 10,0% for concentration 15%w/w pH 1 %(w/w) water solution 6,8

M_w. 42 kDa (for soluble part) particle size 100-500nm

PUR /PAGA Ca/Na ratio (proportion):

PUR 85 parts

PAGA Ca salt 15 parts

Procedure:

500ml Dimethylformamide is added into heating, glass rector with mixer. The cooler is switched on. Content of the vessel is heat up to 100⁰C when mixing. During 120 minutes 150g of polyurethane granulate is added. When dissolved, the solution is cooled down to 60⁰C and then filtrated on the vaccum filter. Filtrate is pumped into the container of the nanofibre machinery. 26,5 g of PAGA Ca salt is then added. When the solution is homogenous, the viscosity and conductivity is adjusted. Nanofibres are applied on the PPSB to create a layer of 20g/m² at voltage of 25kV on electrode and 4IcV on the opposing electrode. The layer of nanofibres is then taken out of PPSB and cut for tapes of dimension 200mm x 15mm. These tapes can be used for "quick stop" bleeding for small wound on the fingers, wrist and ankles etc. The tapes are sufficiently flexible but still solid, so they can be used for wrapping around the wound.

Example No. 9 Hemostatic nanofibre dressing intended to stop bleeding faster than traditional dressings

Raw material:

SloviolR 16% water solution of PVA (supplier CHZNovaky, Slovakia)

Grade of hydrolysis 88%

Saponification 141mgKOH/g

Specific conductivity 31 OμS/cm

Dry matter 15.9%w/w

M_w 85 kDa Polyanhydroglucuronic acid Ca²⁺ salt PAGA (supplier Alltracel Pharma Ltd. Dublin)

Loss on drying 1 ,9%w/w

Carboxyl Content 22,7%w/w

Ca²⁺ Content 8,8% w/w

Na⁺ Content 0, 1 %w/w water solubility 10,0% for concentration 15%w/w pH 1 %(w/w) water solution 6,8

M_w 42 kDa (for soluble part) particle size 100-5 OOnm

PVA /PAGA Ca/Na ratio (proportion):

PVA 60 parts

PAGA Ca/Na salt . 40 parts

Catalyser none

Procedure:

Procedure is the same as in Example No. 1. When the polymer substances are mixed, a catalyser is added.

Haemostatic effects

PAGA Ca/Na promotes blood clotting by activating platelets, stimulating activation of the intrinsic system of blood coagulation and by providing a structural scaffold that acts both physically and as an assembly point for the relevant clotting factors. These effects are predominately driven by surface area effects. The surface area of a nano version of PAGA Ca/Na would have a much greater surface area than a native micronised form. The PVA / PAGA Ca/Na nanofϊbre was added to blood plasma. An equal quantity of PAGA Ca/Na was also added to blood plasma. Over time the activity of the coagulation enzymes was measured (Figure 14).

This resulted in the PVA / PAGA Ca/Na nanofibre having an efficacy of more than five times the micronised form of the PAGA Ca/Na when equal quantities of the products were used by weight. Such an advantageous result could also mean one fifth of the product could be used in nano form to retain the same efficacy. This nano form of the PAGA technology therefore has efficacy and cost benefits.

Wound Healing Benefits

The nanofibre form of PAGA stops bleeding faster than the native micron sized PAGA. As this haemostatic effect is the first stage of wound healing, the nanofibre PAGA format may also have a greater effect on wound healing.

Rats underwent experimental wounding (three round wounds about 1 cm diameter on dorsal region of each animal were performed). The PAGA Ca/Na /PVA nanofibre was topically administered twice a day during the wound healing process. The wound reduction, closure and healing (incidence of wound secretion, haemorrhagic crust and scar formations) were monitored daily. At the end of the healing process all rats were sacrificed and samples of dermal wounds for histological examination were taken.

^■ In both histological analysis and wound diameter measurement the PAGA Ca/Na /PVA nanofibre performed better at stimulating wound healing .that the wound pad that contained an equal quantity of native micron sized PAGA Ca/Na. The nanofibre treated wounds were dry. without inflammatory secretion with formation of low hemorrhagic crusts during the acute healing process demonstrating the advantage of the nanofibre PAGA format. Example No. 10 Wound healing nanoiibre dressing heals faster than traditional dressings

Raw material:

- same as Example No. 1 and 2

Procedure:

The efficiency of PVA / PAGA Ca/Na nanofibre for wound healing was demonstrated on healthy adult rats (strain WISTAR, males, body weights, range 229.7 - 254.Ig) and compared with untreated negative controls and those with normal non nano wound pads. The rats underwent experimental wounding (three round wounds about 1 cm diameter on dorsal region of each animal were performed). The PVA / PAGA Ca/Na nanofibre was topically administered twice a day during the wound healing process. The wound reduction, closure and healing (incidence of wound secretion, haemorrhagic crust and scai' formations) were monitored daily. At the end of the healing process all rats were sacrificed and samples of dermal wounds for histological examination were taken.

Clinical examination of dermal wound healing was performed daily. The size of wounds was measured with a calliper during the healing process. The percentage of wound reduction was calculated using the following formula:

% Wound reduction = Wound area Day 0 - Wound area D X

Day O x 100

X days (an appropriate day of measurement)

The following parameters were evaluated microscopically: Epidermal and dermal regeneration, granulation tissue formation, presence or absence of oedema, congestion, haemorrhase. thrombosis and intravascular or extravascular fibrin formation. Thickness of granulation tissue and epidermal regeneration was scored as follow:

Table 1 - Scoring system

Photography of the wounds was taken in all animals prior to the first administration of the nano wound pad (Day 0) and on Days 2, 4, 6. 8, 10, and further according to the healing process of each individual rat.

Analysis after completion of the trials wound contractions and the healing process was significantly more rapid after in the group where the PVA / PAGA Ca/Na nanofibre administration was performed.

Complete remodelling of epidermis and dermis was found in most rats after the PVA / PAGA Ca/Na nanofibre administration on Day 1 1 , while only moderate epidermal and dermal organisation was observed in the negative controls. Significant improvements in the healing time were found between the PVA / PAGA Ca/Na nanofibre treated animals and the negative control animals.

Anti Adhesion Benefits

Adhesions are scars that form abnormal connections between tissue surfaces. Postsurgical adhesion formation is a natural consequence of surgery, resulting when tissue repairs itself following incision, cauterization, suturing, or other means of trauma. Postsurgical adhesions are very common and complications associated with adhesions are high, and may include:

Small bowel obstruction - 49% to 74% Infertility - 15% to 20% Chronic pelvic pain - 20% to 50%

Adhesion barriers are directly applied to specific sites of surgical trauma to provide a physical barrier that separates traumatized tissue from other tissues during normal healing. - Bioresorbable adhesion barriers help reduce the incidence, extent, and severity .;. of postoperative adhesions. Normally the barriers remain at the sites of placement for up ^':^'• to seven days, maintaining a barrier effect while the body's normal tissue repair takes place. As the repair occurs, the barrier slowly resorbed into the body.

Currently in its native form PAGA can be used to help prevent adhesions in the body. Simply put nano versions of these barriers would perform better. Almost all of human tissues and organs are created in nanofibrous forms or structures for example bone, collagen, cartilage and skin. When the cells involved in the healing process are in contact with nanofibre sized structures they can attach and function normally to repair the damaged area with greater efficiency and less scarring. The nanofibre form of the PAGA technology therefore would have a greater efficacy than the native micro sized PAGA in reducing adhesions. Tissue engineering

Tissue engineering uses of a combination of cells, engineering and materials methods, and suitable biochemical and physio-chemical factors to improve or replace biological functions. Examples include using living fibroblasts in skin replacement or repair, cartilage repaired with living chondrocytes, or other types of cells used in other ways. The cells utilized are normally implanted to the relevant area in artificial scaffolds that are capable of supporting tissue formation. These scaffolds should allow one or more of the following to take place:

Allow cell attachment and migration;

Deliver and retain cells and biochemical factors;

Enable diffusion of vital cell nutrients and expressed products; and/or

Exert certain mechanical and biological influences to modify the behaviour of the cell phase.

A number of different methods have been described in literature for preparing porous ' structures to be employed as tissue engineering scaffolds. Amongst these textiles present .^! the most optimum delivery and variability. Utilising polymers such as collagen, ^Xl polyglycolide and many others have been successfully employed for- the preparation of non-woven meshes.

Utilising PAGA containing nanofϊbres for the creation of these scaffolds provides many advantages over the current scaffolds. The PAGA containing nanofibre scaffolds have a high surface-to-volume ratio which enhances cell adhesion. Cell migration, proliferation, and differentiated function are dependant on adhesion and should be enhanced on nano- fϊbrous scaffolds. These are also enhanced by the presence of PAGA and based on this, nanofibre scaffolds should serve as a better environment for cell attachment, proliferation and function than traditional current scaffolds. The invention is not limited to the embodiments hereinbefore described which may be varied in detail.

References:

1 www.ecmjoumal.org ; www.zapmeta.com

2 Gajanan Bhat and Youneung Lee, „ Recent advancements in Electrospun nanofibres" Proceedings of the twelfth international symposium of Processing and Fabrication of Advanced materials, EdTS Srivatsan &RA Vain, TMS, 2003

3 Shorygin,P.P,and Kheit, E.V.,(1937) Celulose of cellulose with nitric acid and nitrogen peroxide.Zh.Obsh.Khim., 7, 188-92

4 Yackel, E.C. and Kenyon, W.O. (1942)Oxidation of cellulose by nitrogen dioxide. JAm.Chem.Soc., 64, 121-7.

5 M Jelinkova, J Briestensky, Santar, B, Rfhova. In vitro and in vivo immunomodulatory effects of microdispersed oxidized cellulose.: International Immunophaπna- cology 2 (2002) 1429 -1441 ^■

6 J. Rysava et al. Cellulose interaction of oxidized cellulose with fϊbrin(ogen) and blood platelets, Senzore and Actuators B 6903 (2003) 1 - 7, Publisher by Elsevier Science B.V.

7 Won Keun Son, et al., Preparation of Ultrafine Oxidized Cellulose Mats via Electrospinning, Biomacromolecules, S (I)₅197 - 201. 2004.10.102 l/bmO34312g S 1525-7797(03)04312-5. Web Release Date: November 1 1, 2003

Claims

Claims

1. A mesh comprising nanofibers of oxidised polysaccharide and a fibre-forming polymer.

2:. A mesh as claimed in claim 1 wherein the oxidised polysaccharide is uniformly dispersed in the form of molecules and/or nanoparticles in a matrix of the fibre- forming polymer.

3. A mesh as claimed in claims 1 or 2 wherein the oxidised polysaccharide is oxidised glucan.

4. A mesh as claimed in claim 3 wherein the oxidised glucan is polyanliydroglucuronic acid [PAGA] or salts or intermolecular complexes [IJMCS] thereof.

5. A mesh as- claimed in claim 4 wherein the salts are derived from inorganic cations selected from the group: Li⁺, Na⁺, K⁺, Ag⁺ , Mg²⁺ Ca²⁺ , Zn²⁺, Co²⁺, Cu²⁺, Al³⁺, Fe²⁺,Fe³⁺, Ga³⁺ and combinations thereof.

6. A mesh as claimed in any one of claims 1 to 5 wherein the weight ratio of polysaccharide to fibre-forming polymer is from 99:1 to 20:80.

7. A mesh as claimed in claim 6 wherein the ratio is from 80:20 to 60:40.

8. A mesh as claimed in any one of claims 1 to 7 wherein the fibre-forming polymer is substantially soluble in water.

9. A mesh as claimed in claim 8 wherein the fibre-forming polymer soluble in water is polyacrylic acid and their salts and water soluble copolymers thereof. poly(met)acrylamide, polyhydroxypropylmethacrylamide. polyvinylalcohol, salts of chitosane, salts of alginic acid, cellulose derivatives soluble in water, or mixtures thereof.

10. A mesh as claimed in any one of claims 1 to 7 wherein the fibre-forming polymer is subtantially insoluble in water.

U . A mesh as claimed in claim 10 wherein the polymer is polyurethane, polyacrylate, polyesters or cellulose derivatives insoluble in water.

12. A mesh as claimed in any one of claims 1 to 11 wherein the nanofibres have an average diameter in the range of from 50 to 1000 nm.

13. A mesh as claimed in any one of claims 1 to 12 wherein the nanofibres have an average diameter in the range of from 100 to 500 nm.

14. A mesh as claimed in any one of claims 1 to 13 wherein the nanofibres have an average diameter in the range of from 200 to 400 nm.

15. A mesh as claimed in any one of claims 1 to 14 having a specific surface area in the range of from 1 to 100 m /g.

16. A mesh as claimed in any one of claims 1 to 15 having a specific surface area in the range of from 5 to 50 m /g.

17. A mesh as claimed in any one of claims 1 to 16 having an average pore size. of from 10 to 400 i.ni.

18. A mesh as claimed in any one of claims 1 to 17 having an average pore size of from 20 to 80 nm.

19. A mesh as claimed in any one of claims 4 to 18 wherein the polyanhydroglucuronic acid [PAGA] or salt thereof is derived from a starch, cellulose or gum, or is of microbial origin (e.g. polycellobiuronic acid).

20. A mesh as claimed in any one of claims 4 to 19 wherein the PAGA comprises a microdispersed cellulose or derivative thereof.

21. A mesh as claimed in any one of claims 1 to 20 wherein the nanofibres are not crosslinked.

22. A mesh as claimed in any one of claims 1 to 20 wherein the nanofibres are crosslinked.

23. A mesh as claimed in claim 22 wherein crosslinking linkage in the nanofϊbre is created by covalent reactions between reaction groups of oxidised polysaccharide — oxidised polysaccharide or reaction groups of oxidised polysaccharide with reaction groups of fibre-forming polymers.

24. A mesh as claimed 23 wherein crosslinking linkage in the nanofibre is- created by condensation reaction groups such as -COOH, -COOR, -OH, -NH₂, -CONH₂, - CONHNH₂, -CONHNHOC-, -CON₃, catalysers of condensation crosslinking process such as Lewis acids may be used which may be selected from the group of H₂SO₄, HCl, H₃PO₄, NaH₂PO₂, ZnCl₂, TiCl₄ or phosphorus oxides or chlorides.

25. A product comprising a mesh as claimed in any one of claims 1 to 24.

26. A product as claimed in claim 25 comprising a number of layers, at least one of the layers comprising a mesh.

27. A product as claimed in claims 25 or 26 further comprising an agent to alter the flexibility of the mesh.

28. A product as claimed in claim 27 wherein the agent increases the .flexibility of the mesh.

29. A product as claimed in claim 27 wherein the agent decreases the flexibility of the mesh.

30. A product as claimed in any one of claims 27 to 19 wherein the agent comprises a polymer selected from the group comprising polyethylene . glycol ' (PEG); polyethylene oxide (PEO); polyurethane (PUR) and combinations thereof.

31. A product as claimed in any one of claims 25 to 30 further comprising one or more additives.

32. A product as claimed in claim 31 wherein the additives are selected from the ^' group comprising: antimicrobials, antiseptics, antibacterials, antioxidants, vitamins, minerals, healing agents and chemotherapeutics. ■ • :

33. A product as claimed in claim 32 wherein the antimicrobial is one or more selected from the group comprising: copper, silver, cobalt and zinc.

34. A product as claimed in claim 32 wherein the antiseptic is one or more 'selected from the group comprising: peroxides ( such as dibenzoyl peroxide, M₂O₂), iodine (such as iodophores, chiniophon), amines and amides (such as Tego acids, biquanide derivatives), ammonium salts (such as cetyltrimethyl amnonium cation), sulphadiazinates (such as Ag. Zn sulphofodiazinates). thiazine (such as methylene blue) and acridine dyes (such as ethacridinium cation).

35. A product as claimed in claim 32 wherein the antibacterial is one or more selected from the group comprising: chlorohexidine, iodine, chiniofon, gentamicin, bacitracin, neomycin and polymyxin B.

36. A product as claimed in claim 32 wherein the healing agent is one or more selected from the group comprising: hyaluronic acid, aloe vera, tissue engineering actives such as living cells and sacrificial materials such as collagen;

37. A product as claimed in any one of claims claim 31 to 336 wherein the additives are impregnated into the mesh.

38. A product as claimed in any one of claims claim 31 to 36 wherein the additives form a .biocompatible intemiolecular complex (IMC) with the • oxidised polysaccharide.

39. A product as claimed in claim 38 wherein the biocompatible intermolecular ^{■ ■} polymer complex is a complex of:

an anionic component comprising polyanhydroglucuronic acid, .which is a • partially or completely , hydrolysed in a normal and/or oxidative- environment; and

a cationic component comprising one or more of the additives.

40. A product as claimed in any one of claims 25 . to 39 wherein .. the nanofibres ' ^■ contain a modifier and/or an emollient.

i41 . A product as claimed 40 wherein the modifier is selected from the group- of monomer or polymer acids, hydroxyacids. such as tartaric acid, citric acid, malic acid or malonic acid, succinic or maleic acid or their isomer fumaric acid or mixture thereof.

42. A product as claimed 40 wherein the emollient is selected from the group of polyhydroxycompounds and/or aminoalcohols such as glycerol- and polyglycerols, ethylene and propylene glycols and low molecular polymers and copolymers thereof (e.g. poloxamer 407) or aminoalcohols, e.g. mono-, di-, triethanolamirie and mixture thereof.

43. A product as claimed in any one of claims 25 to 42 wherein at least one layer is a carrier layer

44. A product as claimed in claim 43 wherein the carrier layer is a textile.

45. A product as claimed in any one of claims 25 to 44 wherein the product is biodegradable.

46. A product as claimed in any one of claims 25 to 45 wherein the product is a wound dressing.

47. A mesh substantially as described herein.

48. A product substantially as described herein.