RU2842190C2 - Composition for wound dressings - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: group of inventions relates to a composition for breaking down and destroying bacteria in a biofilm and for preventing the formation of a biofilm, comprising a first fibrous component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and any combination thereof; at least one tribasic acid and at least one solubilising acid; wherein at least one tribasic acid is selected from the group consisting of phosphoric acid, citric acid and mixtures thereof, and at least one solubilising acid is supported on at least a portion of the fibrous first component and/or is absorbed into at least a portion of the fibrous first component and wherein the solubilising acid is selected from the group consisting of lactic acid, formic acid, succinic acid, acetic acid, hydrochloric acid and mixtures thereof, also relates to a wound dressing comprising a composition, and to a method for preparing a composition comprising a fibrous first component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and combinations thereof; at least one tribasic acid selected from the group consisting of phosphoric acid, citric acid and mixtures thereof, and at least one solubilising acid, and wherein the solubilising acid is selected from the group consisting of lactic acid, formic acid, succinic acid, acetic acid, hydrochloric acid and mixtures thereof, wherein the method comprises the following steps: a) coating at least a portion of the fibrous first component with a mixture comprising at least one tribasic acid and at least one solubilising acid; and/or b) absorbing, in at least a portion of the fibrous first component, at least one tribasic acid and at least one solubilising acid.

EFFECT: group of inventions provides the improved antimicrobial efficacy in the destruction and destruction of bacteria in the biofilm and in the prevention of the biofilm formation.

25 cl, 10 dwg, 1 tbl, 15 ex

Description

The present invention relates to a composition that can be used as a wound dressing or as part of a wound dressing, and to wound dressings comprising the same. More specifically, the present invention relates to a composition that destroys and kills bacteria in a biofilm and also prevents the formation of a biofilm.

Biofilms are described by Philips PL, et al., Biofilms Made Easy, Wounds International 2010, 1(3) and are summarized herein. A biofilm is any group of microorganisms in which the cells adhere effectively to each other on a surface. A biofilm may typically be a complex microbial community containing bacteria and fungi. The microorganisms are often embedded in a self-produced matrix of extracellular polymeric substances (EPS). Biofilm EPS, also known as slime (although it should be understood that not everything called slime is a biofilm), is a polymeric conglomerate typically composed of extracellular DNA, proteins, and polysaccharides. The EPS matrix can firmly attach the biofilm to living or nonliving surfaces.

Biofilms are known to form on the surfaces of medical devices such as urinary catheters, implants and sutures. This is problematic because they are involved in diseases characterized by underlying bacterial infection and chronic inflammation.

The field of the present invention is principally wound care. Biofilms are commonly found in wounds, but only relatively recently have they been recognized as causing delayed wound healing. It has even been suggested that almost all chronic wounds have biofilm communities on at least part of the wound bed.

Biofilms can form on living or non-living surfaces and can be prevalent in natural, industrial, and hospital settings. In natural settings, microorganisms such as bacteria can attach to surfaces and form biofilms. As bacteria multiply, they become tightly attached to the surface. Once attached, the bacteria secrete EPS to form a protective matrix. This then leads to the formation of small bacterial colonies that form the initial biofilm. Over time, the biofilm can disperse and attach to other parts of the wound bed, forming new biofilm colonies.

Biofilm formation can develop relatively quickly, with a biofilm capable of forming in less than 24 hours.

Biofilms are thought to stimulate a chronic inflammatory response in an attempt to clear the biofilm from the wound. This response results in an abundance of neutrophils and macrophages surrounding the biofilms. These inflammatory cells secrete large amounts of reactive oxygen species (ROS) and proteases (matrix metalloproteinases (MMPs) and elastase). Proteases can help break down the attachment between biofilms and tissue, displacing the biofilm from the wound. However, ROS and proteases also damage normal and healing tissues, proteins, and immune cells. The chronic inflammatory response is not always successful in clearing the biofilm, and it has been suggested that the biofilm itself is involved in the response. By inducing an ineffective inflammatory response, the biofilm protects the microorganisms it contains and increases the production of exudate, which provides a source of nutrition and helps maintain the biofilm.

Currently, one of the most effective methods to reduce the unwanted effects of biofilms is the physical removal of the biofilm, known as debridement. Debridement involves the removal of dead and infected tissue from the wound. However, this process has limitations as no form of debridement can remove all of the biofilm. Consequently, the biofilm has the potential to regenerate within a short period of time. As a result, the patient must often undergo debridement.

Attempts to prevent biofilm re-formation have also been studied. Mostly, these methods use antimicrobials to kill the microorganisms. However, there are some limitations to this method in that antimicrobials can be used in a variety of ways and the patient's sensitivity and allergies must be considered.

Therefore, there is a need to develop improved methods to kill bacteria in biofilm and also prevent biofilm re-formation.

The present invention has been accomplished by considering the above limitations and problems.

According to a first aspect of the present invention, there is provided a solid composition comprising a first fibrous component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and any combination thereof; at least one tribasic acid and at least one dissolving acid; wherein the at least one tribasic acid and at least one dissolving acid are applied to at least a portion of the first component and/or absorbed into at least a portion of the first component.

It has been unexpectedly discovered that a composition comprising a first component, at least one tribasic acid, and at least one dissolving acid is capable of destroying and preventing the formation of a biofilm and has an antimicrobial effect against microorganisms in the biofilm.

The biofilm described herein is preferably a microbial-based biofilm, although the invention is not limited thereto.

The term 'tribasic acid' (which may also be referred to as 'tribasic acid') is used herein to refer to an acid that has three hydrogen ions to donate to a base in an acid-base reaction. In other words, a tribasic molecule has three replaceable hydrogen atoms.

The composition may comprise one or more tribasic acids. Thus, the composition may comprise two, three, four or more tribasic acids. Typically, the composition comprises one tribasic acid.

The tribasic acid may be selected from the group consisting of citric acid, phosphoric acid, or mixtures thereof.

Preferably, the tribasic acid is citric acid.

Tribasic acids can be in the form of granules, flakes, powders or solutions. Typically, tribasic acid is supplied in powder form.

In preparing the composition of the present invention, the tribasic acid is usually in the form of an acidic solution. Such a solution is prepared by dissolving a quantity of the tribasic acid, usually in the form of a powder, in a volume of water and/or a solvent. The solvent may be aqueous or non-aqueous, but is preferably non-aqueous.

The composition may include a mixture of the first component and a tribasic acid. The tribasic acid may be in contact with the first component.

The tribasic acid is absorbed into, or applied to, at least a portion of the first component. Preferably, the tribasic acid is applied to at least a portion of the surface of the first component. More preferably, the tribasic acid is applied to substantially the entire surface of the first component.

Antimicrobials are generally defined as substances that kill or inhibit the growth of microorganisms. It is generally accepted in wound healing that for a substance to be antimicrobial, it must demonstrate a Log4 bacterial kill rate.

The term 'antimicrobial' is used herein to describe an agent or substance capable of demonstrating a Log4 bacterial kill rate within 24 hours. Conversely, the term 'non-antimicrobial' is used herein to refer to an agent or substance that demonstrates less than a Log4 bacterial kill rate within 24 hours.

The first component may be non-antimicrobial.

The ratio of the first component to at least one tribasic acid may be at least 2:1.

The ratio of at least one tribasic acid to at least one dissolving acid is at least 1:1.

The first component may be completely or partially coated with the tribasic acid.

The first component comprises fibers. The fibers may be woven or non-woven. Preferably, the fibers are non-woven. The fibers may be fully or partially coated with a tribasic acid.

Alternatively, the composition may comprise separate parts of the first component and the tribasic acid. For example, the first component in the form of fibers and the tribasic acid in the form of granules, flakes or powder may be located in separate parts of the composition. The separate parts may, for example, be presented in the form of layers. Alternatively, the tribasic acid and the first component may be mixed.

Additionally or alternatively, the tribasic acid may be associated with the carrier material.

The tribasic acid may be absorbed into or supported on a carrier material. The carrier material may act as a carrier for the tribasic acid. In such embodiments, the tribasic acid should not react with or irreversibly bind to the carrier material. The carrier material may include any suitable material that can absorb, receive, or act as a carrier for the tribasic acid. Typical materials include, but are not limited to, polymers such as cellulose, cellulose derivatives (e.g., ethyl cellulose, methyl cellulose, etc.), cotton, alginate, viscose, polypropylene, polyethylene, or any combination of such materials. Preferably, the carrier material is viscose.

Typically, the carrier material is fibrous. In some embodiments, the first component and the carrier material may be mixed together to form a nonwoven article. The first component and the carrier material may be attached or stitched together.

Preferably, however, the composition does not include a carrier material. The tribasic acid is preferably supported on at least a portion of the first component, as explained previously.

The tribasic acid may be supported on the first component and/or the carrier material by any suitable means known in the art.

Typically, a tribasic acid, typically in powder form, is dissolved in water to form an acidic solution. The acidic solution may then be mixed with a solvent. The first component is then mixed with the acidic solution/solvent mixture. The solvent and optionally at least a portion of the water may be removed by evaporation, for example, to form a solid composition of the present invention. Typically, the composition comprises a tribasic acid supported on the first component.

Alternatively, the tribasic acid can be mixed with water and/or solvent as explained above and sprayed onto the first component.

Preferably, the first component is not dissolved in the solvent during the preparation of the composition. Preferably, the first component is insoluble in the acidic solution or acidic solution/solvent mixture.

It has been observed that preparing an initial composition of the first component and the tribasic acid without initially dissolving the first component in a solvent, and/or where the first component is not soluble in the acidic solution/solvent mixture, allows for the effective preparation of a composition having a larger amount of the first component that can be delivered to the wound. This is preferable to using a first component that is initially dissolved in a solvent, since the total amount of the first component available is diluted in the presence of the solvent. The first component is therefore insoluble in water and solvent.

In cases where the first component comprises chitosan fibers, the solvent is preferably a non-aqueous solvent such as isopropyl alcohol.

The tribasic acid is preferably delivered in the form of an acidic solution. The acidic solution (acid in water) may have a concentration of about 5-80%, preferably about 20-60%, and most preferably about 40-50%.

The tribasic acid may be present in the composition in an amount greater than about 2% of the amount of the first component, preferably greater than about 5% of the amount of the first component, more preferably greater than about 10% of the amount of the first component, and most preferably greater than about 25% of the amount of the first component. The tribasic acid may be present in the composition in an amount of at least about 2% of the amount of the first component, preferably at least about 5% of the amount of the first component, more preferably at least about 10% of the amount of the first component, and most preferably at least about 25% of the first component.

The tribasic acid may be present in an amount of about 2-75% of the amount of the first component, preferably about 10-75% of the amount of the first component, more preferably about 20-75% of the first component, and most preferably about 25-60% of the amount of the first component. Good results have been observed with the tribasic acid present in an amount of about 60% of the amount of the first component. The percentage of acid refers to the relative amounts of the tribasic acid compared to the total amount of the first component in the composition. For example, if the total amount of the first component in the composition was 1 g, a composition comprising 20% of the tribasic acid contains 0.2 g of the tribasic acid.

The first component is selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and any combinations thereof.

The term 'derivative' is used herein to denote a compound that is derived from chitosan or chitin after one or more chemical reactions or modifications. The one or more chemical reactions or modifications may include replacement of one or more amino or hydroxyl protons in the chitosan or chitin; or partial deacetylation of the chitin. For example, a chitin derivative may include partially deacetylated chitin, which may have varying percentages of deacetylation, if desired. Typically, partially deacetylated chitin suitable for use in the present invention has a degree of deacetylation of at least about 50%, more typically at least about 75%, and most typically at least about 85%. Also included within the term 'chitosan or chitin derivatives' are reaction products of chitosan or chitin with other compounds. Such reaction products include, but are not limited to, carboxymethyl chitosan, hydroxylbutyl chitin, N-acyl chitosan, O-acyl chitosan, N-alkyl chitosan, O-alkyl chitosan, N-alkylidene chitosan, N-arylidene chitosan, O-sulfonyl chitosan, sulfated chitosan or chitin, phosphorylated chitosan or chitin, nitrated chitosan or chitin, deoxyhalochitosan, alkalichitin, alkalichitosan, or metal chelates of chitosan, organic salts, etc. Derivatives of chitin or chitosan, including those referred to herein, may also contain functional groups bonded thereto through covalent or non-covalent bonds.

Typically, the first component is chitosan or a chitosan derivative. Preferably, the first component is chitosan.

Chitosan is a derivative of shellfish processing solid waste and can be extracted from fungal culture. It is a cationic polymeric material that is insoluble in water. Chitosan is a known hemostatic agent for use in wound dressings. The term 'hemostatic agent' is used herein to refer to any agent that is capable of producing a clot or plug that stops or reduces bleeding when it comes into contact with blood or other body fluids, such as wound exudate, from a physiological target site or wound area of a human or animal.

There are many different types of chitosan that can be used as a material in wound dressings, with different absorption properties. Different types of chitosan may have different molecular weights, different degrees of deacetylation, different arrangements of the β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine monomers, different chiral forms, or they may be obtained from different species or sources (and fungi), or may be processed in different ways during manufacture. Each and all of these different variations of chitosan materials are used for use in the present invention.

The first component chitosan may have a degree of deacetylation of more than 70%, preferably more than 80% and more preferably more than 85%.

Chitosan materials can exhibit gelling properties when in salt form. To produce a chitosan salt, chitosan is usually mixed with an appropriate acid. The gelling properties of chitosan salts make them desirable for use as wound dressing materials.

Typically, the chitosan and/or chitosan derivative are in the form of fibers. Preferably, the fibers are non-woven. The fibers may be of any desired diameter or length and may be formed into a textile article or pad for use.

Typically, the molecular weight of the first component used in the composition of the present invention is less than about 2,000,000, more typically less than about 1,000,000, and even more typically less than about 500,000, and most typically less than about 175,000.

The first component in a 1% acetic acid solution can have a viscosity of more than 150 cP.

The composition of the present invention comprises a dissolving acid.

The term 'solubilizing acid' is used herein to refer to an acid that, when applied to or associated with a first component, makes the first component more soluble in aqueous body fluids.

The composition may comprise one or more solvent acids. The composition may therefore comprise two, three, four or more solvent acids. Typically, the composition comprises one solvent acid.

The dissolving acid may be selected from the group consisting of succinic acid, malic acid, sulfuric acid, acrylic acid, lactic acid, formic acid, acetic acid, hydrochloric acid, nitric acid, and mixtures of any one or more of these.

The dissolving acid may preferably be a monobasic acid.

The term 'monoprotic acid' (which may also be referred to herein as 'monoacid') is used herein to refer to an acid that has one hydrogen ion to donate to a base in an acid-base reaction. In other words, a monoprotic molecule has one replaceable hydrogen atom.

The monobasic acid may be selected from the group consisting of lactic acid, formic acid, acetic acid, hydrochloric acid, nitric acid, and mixtures of any one or more of them.

Preferably, the monobasic acid is lactic acid, acetic acid, or a mixture thereof. Most preferably, the monobasic acid is lactic acid.

The solvent acid may be in the form of granules, powder, or solution. Typically, the solvent acid is in the form of a solution. Such a solution is prepared by dissolving a quantity of the solvent acid in a volume of water and/or solvent.

The composition may include a mixture of a first component, at least one tribasic acid and at least one dissolving acid.

The dissolving acid may be mixed with the tribasic acid and contacted with the first component in the manner described above with respect to the tribasic acid alone. Alternatively, the tribasic and dissolving acids may be contacted with separate portions of the first component and then combined into a composition. For example, when the first component is in the form of fibers, a portion of the fibers may be partially or completely coated with the tribasic acid and a separate portion of the fibers may be partially or completely coated with the dissolving acid.

Typically, a solvent acid is mixed with a tribasic acid and contacted with the first component.

At least one solvent acid or a mixture of at least one solvent acid and at least one tribasic acid may be absorbed into, or coated onto, at least a portion of the first component. Preferably, at least one solvent acid or a mixture of at least one solvent acid and at least one tribasic acid is coated onto substantially the entire surface of the first component. In some embodiments of the invention, the tribasic acid may be absorbed into, or coated onto, at least a portion of the first component, and the solvent acid may then be absorbed into, or coated onto, at least a portion thereof, or vice versa.

Alternatively, the composition may comprise separate parts of the first component, at least one tribasic acid and at least one dissolving acid. For example, the first component is in the form of fibers, and the tribasic and dissolving acids may be in the form of granules, flakes and/or powder, whereby the first component and the acids are located in different parts of the composition. For example, the separate parts may, for example, be in the form of layers. Alternatively, the first component, the tribasic acid and the dissolving acid may be mixed.

In some embodiments of the invention, at least one tribasic acid and at least one solvent acid may be associated with the same or separate carrier materials.

The dissolving acid may be associated with the carrier material in the same manner as described herein with respect to the tribasic acid.

The dissolving acid and/or tribasic acid may be supported on the first component and the carrier material.

The carrier material can be viscose.

At least one dissolving acid may be applied to the first component and the carrier material by the same means as described for the tribasic acid.

The first component may be partially or completely coated with a dissolving acid shell.

The dissolving acid is preferably supplied in the form of an acidic solution. The acidic solution (acid in water) may have a concentration of at least about 40%, preferably at least about 60%, and most preferably at least about 80%.

At least one dissolving acid may be present in an amount greater than about 2% of the first component, preferably greater than 5% of the first component, and more preferably greater than about 10% of the first component. At least one dissolving acid may be present in the composition in an amount of at least about 2% of the first component, preferably at least about 5% of the first component, more preferably at least about 10% of the first component, and most preferably at least about 25% of the first component.

At least one solvent acid may be present in an amount of about 2-50% of the amount of the first component, preferably about 10-40% of the amount of the first component, or more preferably about 20-30% of the amount of the first component. Preferably, at least one solvent acid may be present in an amount of about 25% of the amount of the first component. In the same way as for the tribasic acid, the percentages refer to the relative amounts of the solvent acid compared to the total amount of the first component.

Without the presence of a solvent acid, it was observed that a higher amount of tribasic acid in the composition resulted in greater effectiveness against microorganisms. Such a composition may include tribasic acid in an amount of at least about 25%, preferably at least about 35%, more preferably at least about 50%, and most preferably at least about 60% of the amount of the first component.

However, it has been found in studies that a composition having a solvent acid content of about 15-30% of the amount of the first component can reduce the amount of tribasic acid required to provide the desired effect. For example, it has been shown that a composition having a tribasic acid content of more than 20% gives comparable results relative to a composition having a solvent acid content of 15-20%, but a reduced amount of tribasic acid of about 5% or less.

Therefore, an embodiment of the present invention includes a composition comprising a first component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and combinations thereof; 20-75% of at least one tribasic acid and 10-40% of at least one dissolving acid.

Good results were observed for a composition having a solvent acid content of 20-35%, especially 25-30%, of the first component and a tribasic acid content of 25-45%, especially 30-40%, of the first component.

The combined acid may be present in the composition in an amount of at least about 4% of the amount of the first component, preferably at least about 10% of the amount of the first component, more preferably at least about 25% of the amount of the first component, and most preferably at least about 45% of the amount of the first component.

The composition of the present invention may be in the form of fibers. The fibers may be woven or non-woven. Preferably, the fibers are non-woven.

Typically, the composition is in the form of fibers. More typically, the composition is in the form of non-woven fibers or non-woven textiles.

When used, the composition may be converted into a gel, hydrogel or liquid. In such forms, the composition may be used without further modification (for example, it may be applied directly to a wound) or the composition may be applied to a delivery vehicle.

The hydrogel is obtained by preparing a solution of a tribasic acid and a dissolving acid in water, optionally mixing the solution with a solvent and adding the first component to the solution. The first component absorbs the solution and the gel. The degree of gelation and the strength of the gel can be varied by the amount of the first component added to the solution. The greater the amount of the first component in the solution, the denser the gel and vice versa. The gel can then be dried to varying degrees depending on the form of the desired composition.

Alternatively, the hydrogel is prepared by obtaining the composition of the present invention in dry form and mixing with water. The composition absorbs water and gel. Also, the degree of gelation and the density of the gel can vary from the amount of the composition mixed with water, or from the amount of water mixed with the composition. The greater the amount of the composition relative to water, the denser the gel and vice versa. Then the gel can be dried to varying degrees depending on the form of the desired composition.

For example, a hydrogel sheet can be produced by transferring a hydrogel into a template such as a cuvette and drying the hydrogel to an extent that does not remove all the water but leaves enough water to form a gel.

Alternatively, a dry film can be produced by transferring the hydrogel into a template, such as a cuvette, and drying the hydrogel to such an extent as to remove substantially all of the water. The resulting composition is in the form of a dry film. The film is preferably flexible.

The delivery means may include polyurethane foam, polyurethane film, woven textile, superabsorbent material, a medical device such as a catheter, stent, and the like. The delivery means may include more than one of each of the above components and/or may include a combination of one or more of the above.

For example, the delivery mechanism may include one or more polyurethane foams and a polyurethane film. Alternatively, the delivery mechanism may include a woven viscose textile.

The term 'superabsorbent material' is used herein to refer to a hydrophilic material which is water swellable but not water soluble and which is capable of absorbing liquid to a level greater than 2000% with a liquid retention of greater than 85%. Preferably, the superabsorbent material is capable of absorbing liquid to a level greater than 2500% with a liquid retention of greater than 90%.

The term 'water-swellable' is used herein to refer to a material which, when in contact with water or a liquid containing water, will absorb the liquid and swell, but is substantially insoluble in such liquid.

The term 'water-soluble' is used herein to refer to a material which, when in contact with water or a liquid containing water, dissolves in such liquid.

The superabsorbent material can be selected from polymeric materials such as poly(vinyl) alcohol (PVA), poly(ethylene oxide) (PEO) and poly(acrylic acid). The superabsorbent material can be chemically modified. For example, the superabsorbent material can be a polymeric material obtained by graft polymerization of acrylic acid on a carboxymethyl cellulose chain. The superabsorbent material can include a chemically modified material selected from starch, cellulose and polymeric materials such as poly(vinyl alcohol) (PVA), poly(ethylene oxide) (PEO), and poly(acrylic acid). The poly(acrylic acid) can be partially neutralized, slightly cross-linked poly(acrylic acid).

The terms "cross-linked" and "cross-linked" are used herein to refer to two or more polymer chains linked by a primary bond, such as a covalent bond. The term "slightly cross-linked" is used herein to refer to embodiments of the invention wherein the number of cross-linking primary bonds in the superabsorbent material is less than the total number of possible cross-linking bonds.

In some embodiments of the invention, the superabsorbent material is selected from polymeric materials such as PVA, PEO, and poly(acrylic acid), preferably partially neutralized, slightly cross-linked poly(acrylic acid). Typically, the superabsorbent material is partially neutralized, slightly cross-linked poly(acrylic acid).

The superabsorbent material may be in the form of fibers. Typically, the superabsorbent material is in the form of nonwoven fibers. The length of the fibers can be up to 100 mm, and is typically from 20-75 mm, more typically from 32 to 51 mm.

The delivery vehicle may be a wound dressing known in the art.

The composition of the present invention can be applied to the delivery vehicle by any suitable means known in the art.

The composition of the present invention may include a first fibrous component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and combinations thereof; and at least one tribasic acid and at least one solvent acid, wherein the first component is substantially partially coated with a shell of the tribasic acid and the solvent acid.

Typically, the composition comprises a fibrous chitosan first component at least partially coated with a shell of a tribasic acid and a dissolving acid. Preferably, the composition comprises a non-woven fibrous chitosan first component at least partially coated with a shell of citric acid and lactic acid. Preferably, the composition further comprises a non-woven fibrous chitosan first component substantially completely coated with a shell of a mixture of citric acid and lactic acid.

According to a further aspect of the present invention, there is provided a wound dressing comprising a composition as described herein.

The wound dressing may be a wound dressing that forms a gel when in contact with a liquid, such as wound exudate. In the field of gelling wound dressings that aim to stop bleeding from a bleeding wound, the composition of the present invention is particularly surprising, since the tribasic acid on non-woven wound dressings does not tend to gel and is known to exhibit negative effects on water absorption and retention.

The wound dressing may include a composition as described herein and a delivery vehicle as described herein. The composition may be applied to the delivery vehicle by a shell or the like. The composition and the delivery vehicle may form a multilayer wound dressing.

The composition of the present invention or a wound dressing comprising such a composition may also comprise additional components. Such additional components include, but are not limited to, antimicrobial agents; pharmaceuticals; complexing agents; moisturizing agents such as surfactants; growth factors; cytokines; agents that absorb agents that slow down healing such as MMPs (matrix metalloproteinases) and elastase; and/or other components of wound dressings such as calcium, vitamin K, fibrinogen, thrombin, factor VII, factor VIII, clays such as kaolin, oxidized regenerated cellulose, gelatin or collagen, etc.

Suitable antimicrobial agents may be selected from a list including silver, polyhexamethylene biguanide (PHMB), iodine, octenidine, copper, chlorhexidine gluconate (CHG), miconazole, metronidazole, and combinations of one or more of these.

The antimicrobial agent may be applied to or absorbed into the first component and/or carrier material in the same manner as described herein with respect to the tribasic and/or dissolving acids.

The composition of the present invention may be mixed with other compositions useful in wound care. In some embodiments, the composition of the present invention may be mixed or blended with one or more hemostatic agents. The hemostatic agents may be in the form of fibers, granules, flakes, powder, or any combination thereof. Preferably, the hemostatic agent is in the form of granules.

For example, the composition of the present invention, as described herein, can be mixed with a hemostatic agent, such as Celox® granules, a commercially available chitosan-based hemostatic agent.

According to a further aspect of the present invention, there is provided a composition as described herein for use as a therapeutic agent.

According to a further aspect of the present invention, there is provided a composition as described herein for use in the treatment of wounds.

According to a further aspect of the present invention, there is provided a composition as described herein for use in disrupting and killing bacteria in a biofilm.

According to a further aspect of the present invention, there is provided a composition as described herein for use in the prevention of biofilm formation.

According to a further aspect of the present invention, there is provided a method for producing a composition comprising a first fibrous component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and combinations thereof; at least one tribasic acid and at least one dissolving acid, the method comprising the steps of:

(a) coating at least a portion of the first component with a mixture comprising at least one tribasic acid and at least one dissolving acid; and/or

(b) absorbing into at least one portion of the first fibrous component a mixture comprising at least one tribasic acid and at least one solvent acid.

According to a further aspect of the present invention, there is provided a method for producing a composition comprising a first fibrous component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and combinations thereof; at least one tribasic acid and at least one dissolving acid, the method comprising the steps of:

(a) mixing the tribasic acid with water and/or a solvent to obtain a tribasic acid solution;

(b) mixing a tribasic acid solution with a solvent acid solution obtained by mixing a solvent acid with water and/or a solvent to obtain a mixed acid solution;

(c) optionally mixing the mixed acid solution with a solvent; and

(d) adding a first fibrous component to the solution obtained in step (c).

The method may further comprise the step (e) of drying the mixture obtained in step (d). The drying step may remove some or all of the solvent and/or water in the composition.

For compositions where the first component comprises chitosan fibers, the solvent of steps (a)-(c) is typically non-aqueous.

Additional aspects of the present invention may include any or all of the features described in relation to the first aspect of the present invention, if desired or necessary.

Embodiments of the present invention will now be further described with reference to the following non-limiting examples and accompanying drawings, in which:

Fig. 1: is a graph showing the results of testing using the MBEC assay for Pseudomonas aeruginosa ATCC 13359;

Fig. 2: is a graph showing the test results using the MBEC assay for Staphylococcus haemolyticus ;

Fig. 3: is a graph showing the test results using the MBEC assay for MRSA 308;

Fig. 4: is a graph showing the results of testing using the CDC reactor model for Pseudomonas aeruginosa ATCC 10434;

Fig. 5: is a graph showing the results of testing using the CDC reactor model for Staphylococcus haemolyticus NCTC 11042;

Fig. 6: is a graph showing the results of testing using the CDC reactor model for methicillin-resistant Staphylococcus aureus 308;

Fig. 7: is a graph showing the results of testing using the CDC reactor model for Pseudomonas aeruginosa ATCC 10434;

Fig. 8: is a graph showing the results of testing using the CDC reactor model for Pseudomonas aeruginosa ATCC 10434;

Fig. 9: is a graph showing the results of testing using the CDC reactor model for Staphylococcus aureus ;

Fig. 10: is a graph showing the results of testing using the CDC reactor model for Staphylococcus aureus .

General methods for obtaining a sample

General Method 1: First Component and Tribasic Acid

A tribasic acid powder (e.g., citric acid) was dissolved in deionized water and then mixed with a non-aqueous solvent (e.g., IPA). The first component, usually in the form of non-woven fibers, was placed in the tribasic acid solution and allowed to absorb the solution. The solution was then dried using a thermal dryer, leaving behind solid chitosan, chitin, or derivatives thereof coated with the tribasic acid.

General Method 2: First Component with Tribasic Acid and Monobasic Acid

A tribasic acid powder (e.g., citric acid) was dissolved in deionized water and then mixed with a solution of a solvent acid (e.g., lactic acid). This was then mixed with a non-aqueous solvent (e.g., IPA). The first component, usually in the form of non-woven fibers, was placed in the mixed acid solution and allowed to absorb the solution. The solution was then dried using a thermal dryer to leave a solid chitosan, chitin, or derivative thereof coated with a mixture of the tribasic acid and the solvent acid.

Sample compositions

The following are examples of compositions obtained in accordance with the present invention.

Example 1 (control):

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.439 g) to obtain a nominal 32.5% composition.

Example 2 (control):

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.027-0.068 g) to obtain a nominal 2-5% composition.

Example 3 (control):

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.27-0.41 g) to obtain a nominal 20-30% composition.

Example 4 (control):

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.81-0.94 g) to obtain a nominal 60-70% composition.

Example 5:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.027-0.068 g) and lactic acid (0.34 g) to obtain a nominal composition of 2-5% tribasic acid and 25% monobasic acid.

Example 6:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.27-0.41 g) and lactic acid (0.34 g) to obtain a nominal composition of 20-30% tribasic acid and 25% monobasic acid.

Example 7:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.81-0.94 g) and lactic acid (0.41 g) to obtain a nominal composition of 60-70% tribasic acid and 30% monobasic acid.

Example 8:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.04 g) and lactic acid (0.2 g) to obtain a nominal composition of 3% tribasic acid and 15% monobasic acid.

Example 9:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.34 g) and lactic acid (0.2 g) to obtain a nominal composition of 25% tribasic acid and 15% monobasic acid.

Example 10:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.81 g) and lactic acid (0.27 g) to obtain a nominal composition of 60% tribasic acid and 20% monobasic acid.

Example 11:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.41 g) and lactic acid (0.34 g) to obtain a nominal composition of 30% tribasic acid and 25% monobasic acid.

Example 12:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.54 g) and lactic acid (0.34 g) to obtain a nominal composition of 40% tribasic acid and 25% monobasic acid.

Example 13:

Non-woven fibers of 100% chitosan (1.35 g) were coated with citric acid (0.54 g) and lactic acid (0.41 g) to obtain a nominal composition of 40% tribasic acid and 30% monobasic acid.

Examples of wound dressings

Example 14 (control):

A composition of the present invention was prepared containing nominally 2 g of 100% chitosan nonwoven fibers in 8 g of deionized water with 0.6 g of citric acid. The composition was applied to the surface of a non-woven polyurethane foam and allowed to dry. The foam containing the dry composition of the present invention was then adhesively bonded to the polyurethane foam, distal to the bonded layer of the polyurethane film, to form a wound contact layer.

Example 15 (control):

A composition of the present invention was prepared containing nominally 2 g of 100% chitosan nonwoven fibers in 8 g of deionized water with 0.6 g of citric acid. The composition was applied to the surface of a viscose woven textile and allowed to dry.

MBEC Analysis 1

To determine the efficacy of antimicrobial agents against biofilms of various microorganisms, the MBEC (minimum biofilm eradication concentration) assay was used.

The MBEC assay is a high yield screening assay used to determine the efficacy of antimicrobials against biofilms of various microorganisms. The MBEC Biofilm Inoculator consists of a plastic lid with 96 pins and a matching base. There are two types of bases that can be used with the MBEC lid. One base contains 96 individual wells. The individual wells allow multiple microorganisms to grow on the same lid pins. The other type of base is a profiled through base that can only contain a single microorganism. Biofilms are mounted on the pins under batch conditions (no nutrient flow into or out of an individual well) with gentle mixing. The resulting biofilm is transferred to a new 96 well plate for antimicrobial efficacy testing. The assay design allows for the simultaneous testing of multiple biocides at multiple concentrations with replicate samples, making this an effective screening tool.

Tested microorganisms

Pseudomonas aeruginosa ATCC 13359

Staphylococcus haemolyticus

MRSA 308

Comparative test samples

Control: Phosphate buffered saline (PBS)

Sample A: 100% chitosan non-woven fibers with Ag (nominal 1%) and lactic acid to yield a nominal composition of 25% monobasic acid

Sample B: Non-woven fibres of carboxymethylated cellulose with Ag (nominal 1%) (Aquacel Ag®)

Sample C: 100% Chitosan Non-Woven Fibers with Lactic Acid to Achieve a Nominal Composition of 25% Monobasic Acid

Sample D: 100% Chitosan Non-Woven Fibers with Acetic Acid to Obtain a Nominal Composition of 25% Monobasic Acid

Sample E: 100% Chitosan Non-Woven Fibers with Citric Acid to Achieve a Nominal Composition of 25% Tribasic Acid

Obtaining bacterial inoculum

A 24-hour culture of each organism was collected from a tryptone soy agar (TSA) plate or a brain heart infusion agar (BHIA) plate and suspended in either 20 ml tryptone soy broth (TSB) or 20 ml brain heart infusion broth (BHIB). The resulting bacterial suspension was diluted to obtain an initial OD590 of 0.10±0.03, which corresponds to a bacterial concentration of 108 CFU ml ^-1 . This initial inoculum was serially diluted an additional six times to obtain progressively lower bacterial loads (i.e., 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ CFU ml ^-1 ). The initial bacterial concentration for each organism was typically 1×10 ⁸ ±5×10 ⁷ CFU ml ^-1 .

MBEC Analysis

Biofilm of each microorganism was grown on pin projections on the lid of a microtiter plate for 48 hours at 37°C, 50 rpm. After 48 hours, the pin lids were removed, rinsed briefly in phosphate buffered saline (PBS) to remove planktonic bacteria, and then placed in an agent selection plate for 24 hours. To prepare the wound dressing selection plate, 1 ^cm2 pieces were cut using sterile scissors and placed in the designated wells of the microtiter plate. The agent bead test selection plate was prepared by weighing 30 mg±3 mg of each bead composition into the wells of the microtiter plate. The wound dressings and beads were then activated with 150 μl PBS. After treatment, the lid pin projections were washed twice in PBS, then transferred to 200 μl neutralizer and placed in an ultrasonic water bath for 5 min to recover any remaining adherent bacteria. Serial dilutions were performed on the resulting reconstituted medium and drop plates were used to quantify the recovered bacteria. All samples were tested in triplicate unless otherwise stated.

The results are shown in Table 1 and Figs. 1-3. The results in Figs. 1-3 refer to samples B, C and E.

It is evident from the graphs shown in Figs. 1-3 that sample E, comprising chitosan coated with a tribasic acid, is effective against all three tested microorganisms. Sample C, comprising chitosan fibers coated with a monobasic acid, demonstrated microorganism concentrations for all three tested microorganisms. Finally, sample B, comprising carboxymethylated cellulose with silver, showed good results against Staphylococcus haemolyticus , but was not effective against either Pseudomonas aeruginosa ATCC 13359 or MRSA 308.

Sample Organisms P. aeruginosa
(1 strain) P. aeruginosa
(2 strain) E. faecillis (VRE) MRSA S. epidermidis S. haemolyticus A Comparable to untreated control Comparable to untreated control >Log 4 Varies between Log2 and > log 4 >Log 4 >Log 4 B ≤Log 3 Comparable to untreated control Comparable to untreated control Varies between Log2 and > log 4 Log 2 >Log 4 C Comparable to untreated control ≤Log 2 >Log 4 Varies between Log2 and > log 4 >Log 4 >Log 4 D >Log 4 ≤Log 3 >Log 4 Varies between Log2 and > log 4 >Log 4 ≤Log 2 E >Log 4 >Log 4 >Log 4 >Log 4 >Log 4 >Log 4

Table 1: Test results after MBEC analysis

Reactor model CDC 1

To determine the biofilm removal capacity of seven wound dressings against three bacterial samples using the CDC reactor method.

Tested microorganisms

Staphylococcus haemolyticus NCTC 11042

Pseudomonas aeruginosa ATCC 10434

Methicillin-resistant Staphylococcus aureus 308

Test samples

Control: phosphate buffered saline (PBS)

Sample F: 100% chitosan non-woven fibers with 15% lactic acid and 3% citric acid. This corresponds to 1.35 g chitosan, 0.2 g lactic acid and 0.04 g citric acid (example 8).

Sample G: 100% chitosan non-woven fibers with 15% lactic acid and 25% citric acid. This corresponds to 1.35 g chitosan, 0.2 g lactic acid and 0.34 g citric acid (example 9).

Sample H: 100% chitosan non-woven fibers with 20% lactic acid and 60% citric acid. This corresponds to 1.35 g chitosan, 0.27 g lactic acid and 0.81 g citric acid (Example 10).

Sample I: 100% chitosan non-woven fibers with 3% citric acid. This corresponds to 1.35 g chitosan and 0.04 g citric acid (example 2).

Sample J: 100% chitosan non-woven fibers with 25% citric acid. This corresponds to 1.35 g chitosan and 0.34 g citric acid (example 3).

Sample K: 100% chitosan non-woven fibres with 60% citric acid. This corresponds to 1.35 g chitosan and 0.81 g citric acid (example 4).

Samples were obtained following the methods described previously.

Dressing samples were cut into pieces of approximately 1.5 ^cm2 prior to use. Phosphate buffered saline (PBS) was used as a control.

Obtaining bacterial inoculum

A 24-hour culture of the test bacteria was harvested from a tryptone soy agar (TSA) plate using a sterile sponge and resuspended in 20 ml tryptone soy broth (TSB). The bacterial suspension was diluted to obtain an OD590 of 0.10 ± 0.03, which corresponds to a bacterial concentration of 10 ⁸ ± 5 × 10 ⁷ CFU ml ^-1 . It was further diluted in TSB and used as an inoculum for the CDC reactor containing the test samples. The CDC reactor was incubated for 48 hours at 37 °C, shaking at 50 rpm to stimulate biofilm growth.

Biofilm treatment

After 48 hours, challenge samples were removed from the CDC reactor and washed 3 times in sterile PBS to remove planktonic bacteria. Washed samples were then processed by sandwiching the sample between two 1.5 ^cm2 dressing samples. Dressings were activated prior to testing by adding 350 μl PBS (75% saturation) to each 1.5 ^cm2 piece. Control samples were immersed in 2 ml PBS for P. aeruginosa (or 2 ml PBS+0.1% TSB for S. haemolyticus and MRSA). All samples were tested in triplicate. Organisms were recovered from samples after 24 hours of treatment and quantified using serial dilutions and drop plates.

The results are shown in Fig. 4-6.

It is evident from the graphs shown in Figs. 4-6 that samples G, H, and K were effective against all three microorganisms tested. Samples G and H comprise chitosan fibers coated with a tribasic acid and a monobasic acid. Sample K comprises chitosan fibers coated with a higher amount of tribasic acid. While samples F, I, and J were effective against methicillin-resistant Staphylococcus aureus 308, they were less effective against both Staphylococcus haemolyticus NCTC 11042 and Pseudomonas aeruginosa ATCC 10434.

The results of the CDC reactor model showed that chitosan coated with increasing amounts of tribasic acid, for example, about 60%, was more effective against microorganisms than lower amounts of about 25% or less. Also, the results show that incorporation of a monobasic acid with the tribasic acid can be effective in reducing the amount of tribasic acid required, for example, reducing the amount of tribasic acid to about 25%.

Reactor model CDC 2

To determine the biofilm removal capacity of six wound dressings against two bacterial samples using the CDC reactor method.

Tested microorganisms

Staphylococcus haemolyticus NCTC 8325

Pseudomonas aeruginosa ATCC 10434

Test samples

Control: phosphate buffered saline (PBS)

Sample L: 100% chitosan non-woven fibers with 25% lactic acid and 30% citric acid. This corresponds to 1.35 g chitosan, 0.34 g lactic acid and 0.41 g citric acid (sample 11).

Sample M: 100% chitosan non-woven fibers with 25% lactic acid and 40% citric acid. This corresponds to 1.35 g chitosan, 0.34 g lactic acid and 0.54 g citric acid (Example 12).

Sample N: 100% chitosan non-woven fibers with 30% lactic acid and 40% citric acid. This corresponds to 1.35 g chitosan, 0.41 g lactic acid and 0.54 g citric acid (Example 13).

Example O: 100% Chitosan Non-woven Fibers.

Sample P: 55% chitosan fiber/45% viscose non-woven fiber with 25% lactic acid. This corresponds to 0.74 g chitosan fiber, 0.61 g viscose fiber and 0.34 g lactic acid.

Sample Q: Non-woven carboxymethyl cellulose with ionic silver containing anti-biofilm composition.

Samples were obtained following the methods described previously.

Dressing samples were cut into approximately 1.5 ^cm2 pieces prior to use. Phosphate buffered saline (PBS) was used as a control.

Obtaining bacterial inoculum

A 24-hour culture of each organism was harvested from a tryptone soy agar (TSA) plate and resuspended in 20 ml tryptone soy broth (TSB). The resulting bacterial suspension was diluted to give an initial OD590 of 0.10 ± 0.03, corresponding to a bacterial concentration of 10 ⁸ ± 5 × 10 ⁷ CFU ml ^-1 . This was further diluted to approximately 10 ⁷ CFU ml ^-1 in TSB and used as the initial inoculum for the CDC reactor. The CDC reactor was incubated for 24 and 72 h at 37 °C with shaking at 50 rpm to facilitate biofilm growth.

Biofilm treatment

After 24 and 72 hours, test samples were removed from the CDC reactor and washed 3 times in sterile phosphate buffered saline (PBS) to remove planktonic bacteria. Washed samples were then processed by sandwiching each sample between two pieces of wound dressing material. Dressings were activated prior to testing by the addition of 350 μL of PBS containing 1% TSB. Control samples were immersed in 2 mL of PBS containing 1% TSB. Following the 24 hour treatment period, samples were placed in 2 mL of neutralizer and sonicated for 15 min to recover any remaining adherent bacteria. Serial dilutions of the resulting reconstituted medium were made and drop plates were used to quantify the recovered bacteria. All samples were tested in triplicate.

The results are shown in Fig. 7-10.

Figure 7 shows the number of viable Pseudomonas aeruginosa recovered from preformed 24-hour biofilms after 24 hours of treatment with wound dressing samples. The control was treated with PBS+1% TSB.

Figure 8 shows the number of viable Pseudomonas aeruginosa recovered from preformed 72-hour biofilms after 24-hour treatment with wound dressing samples. The control was treated with PBS+1% TSB.

Figure 9 shows the number of viable Staphylococcus aureus recovered from preformed 24-hour biofilms after 24 hours of treatment with wound dressing samples. The control was treated with PBS+1% TSB.

Figure 10 shows the number of viable Staphylococcus aureus recovered from preformed 72-hour biofilms after 24-hour treatment with wound dressing samples. Controls were treated with PBS+1% TSB.

It is evident from the graphs shown in Figs. 7-10 that samples L, M and N of the present invention are effective against both tested microorganisms. While samples Q and P were effective against Staphylococcus aureus , they were less effective against Pseudomonas aeruginosa ATCC 10434.

It should, of course, be understood that the present invention is not intended to be limited to the above examples, which are described by way of example only.

Claims (27)

1. A composition for the destruction and elimination of bacteria in a biofilm and for preventing the formation of a biofilm, comprising a fibrous first component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and any combination thereof; at least one tribasic acid and at least one solubilizing acid; wherein at least one tribasic acid is selected from the group consisting of phosphoric acid, citric acid and mixtures thereof, and at least one solubilizing acid is applied to at least a portion of the fibrous first component and/or absorbed into at least a portion of the fibrous first component and wherein the solubilizing acid is selected from the group consisting of lactic acid, formic acid, succinic acid, acetic acid, hydrochloric acid and mixtures thereof. 2. The composition of claim 1, wherein the fibrous first component is non-antimicrobial. 3. The composition according to any one of claims 1 or 2, wherein the weight ratio of the fibrous first component and the at least one tribasic acid is at least 2:1. 4. A composition according to any one of the preceding claims, wherein the weight ratio of the at least one tribasic acid and the at least one solubilizing acid is at least 1:1. 5. A composition according to any one of the preceding claims, wherein the fibrous first component is chitosan. 6. The composition of claim 5, wherein the fibrous first component has a degree of deacetylation of at least 70%. 7. The composition according to claim 5 or 6, wherein the fibrous first component has a viscosity of more than 150 cP in a 1% acetic acid solution. 8. A composition according to any one of the preceding claims, wherein the tribasic acid is citric acid. 9. The composition according to any one of the preceding claims, wherein the fibrous first component comprises non-woven fibers. 10. A composition according to any one of the preceding claims, wherein the tribasic acid is present in an amount of 2-75% by weight of the amount of the fibrous first component. 11. The composition according to claim 10, wherein the tribasic acid is present in an amount of 25-60 wt.% of the amount of the first component. 12. A composition according to any one of the preceding claims, wherein the solubilizing acid and the tribasic acid are mixed prior to contact with the fibrous first component. 13. A composition according to any one of the preceding claims, wherein the solubilizing acid and the tribasic acid are applied to the fibrous first component. 14. A composition according to any one of the preceding claims, wherein the solubilizing acid is present in an amount of 2-50% by weight of the amount of the fibrous first component. 15. The composition according to claim 14, wherein the solubilizing acid is present in an amount of 15-20% by weight of the amount of the fibrous first component. 16. A composition according to any one of the preceding claims, further comprising a tribasic acid and/or a solubilizing acid supported on a carrier material. 17. The composition according to claim 16, wherein the carrier material is viscose. 18. The composition according to any one of the preceding claims, further comprising an additional component selected from the group consisting of antimicrobial agents; pharmaceuticals; chelating agents; moisturizing agents; growth factors; cytokines; agents that absorb agents that slow down healing, such as MMP (matrix metalloproteinases) and elastase; calcium; vitamin K; fibrinogen; thrombin; factor VII; factor VIII; clays; oxidized regenerated cellulose; gelatin and collagen. 19. The composition of claim 18, wherein the antimicrobial agents are selected from the group consisting of silver, polyhexamethylene biguanide (PHMB), iodine, octenidine, copper, chlorhexidine gluconate (CHG), miconazole, metronidazole, and combinations of any one or more thereof. 20. A wound dressing comprising a composition as claimed in any preceding claim. 21. A composition according to any one of claims 1-19 for use as a therapeutic agent. 22. A composition according to any one of claims 1-19 for use in the treatment of wounds. 23. A composition according to any one of claims 1-19 for use in the destruction and elimination of bacteria in a biofilm. 24. The composition according to any one of claims 1-19 for use in preventing biofilm formation. 25. A method for producing a composition comprising a fibrous first component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives and combinations thereof; at least one tribasic acid selected from the group consisting of phosphoric acid, citric acid and mixtures thereof, and at least one solubilizing acid, and where the solubilizing acid is selected from the group consisting of lactic acid, formic acid, succinic acid, acetic acid, hydrochloric acid and mixtures thereof, the method comprising the steps of: a) coating at least a portion of the fibrous first component with a mixture comprising at least one tribasic acid and at least one solubilizing acid; and/or b) absorbing into at least a portion of the fibrous first component at least one tribasic acid and at least one solubilizing acid.