RU2775940C2 - Composition for wound bandages - Google Patents

Abstract

FIELD: medicine; medicinal products.

SUBSTANCE: wound bandage including a solid composition is used for surgical treatment of a wound. The solid composition includes the first component, which is chitosan, and tribasic acid, which is citric acid. At the same time, citric acid is present in the amount of at least 10 wt.% of the amount of the first component. Citric acid is applied to at least part of the first component and/or is absorbed in at least part of the first component.

EFFECT: inventions provide elimination of bacteria in biofilm and prevent repeated formation of biofilm.

22 cl, 12 dwg, 2 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 wound dressings, including such. More specifically, the present invention relates to a composition that breaks down and kills bacteria in a biofilm and also prevents the formation of a biofilm.

A description of biofilms is provided by Philips PL, et al., Biofilms Made Easy, Wounds International 2010, 1(3) and is summarized herein. A biofilm is any group of microorganisms in which cells effectively adhere to each other on surfaces. A biofilm can typically be a complex microbial community containing bacteria and fungi. Microorganisms are often embedded in a self-producing matrix of extracellular polymeric substances (EPS). EPS biofilms, also referred to as mucus (although it must be understood that not everything referred to as mucus is a biofilm), is a polymeric conglomerate, usually composed of extracellular DNA, proteins, and polysaccharides. The EPS matrix can firmly attach the biofilm to living or non-living surfaces.

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

The scope of the present invention is fundamentally 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 a portion of the wound bed.

Biofilms can form on living or non-living surfaces and can predominate in natural, industrial and hospital environments. Under natural conditions, microorganisms such as bacteria can attach to surfaces and form biofilms. When bacteria multiply, they become tightly attached to surfaces. Once attached, the bacteria secrete EPS to form a protective matrix. This then leads to the formation of small bacterial colonies forming the original 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 being able to form in less than 24 hours.

It is believed that biofilms 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. Such inflammatory cells secrete large amounts of reactive oxygen species (ROS) and proteases (matrix metalloproteinases (MMPs) and elastase). Proteases can help break the attachment between biofilms and tissue, expelling 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 removing the biofilm, and it has been suggested that the biofilm is involved in the response. By inducing an ineffective inflammatory response, the biofilm protects the microorganisms it contains and increases exudate production, which provides a source of nutrition and helps to maintain the biofilm.

Currently, one of the most effective methods for reducing the unwanted effects of biofilms is the physical removal of the biofilm, known as surgical 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. Therefore, the biofilm has the ability to recover within a short period of time. As a result, the patient often has to undergo surgical debridement.

Efforts to prevent re-formation of the biofilm have also been studied. Preferably, such methods use antimicrobial agents to kill microorganisms. However, there are some limitations to this method in that antimicrobials can be used in a variety of ways and patient sensitivity and allergy must be considered.

Therefore, there is a need to develop improved methods for killing bacteria in a biofilm and also preventing re-formation of the biofilm.

The present invention has been made in consideration of the above limitations and problems.

According to a first aspect of the present invention, a solid composition is provided comprising a first component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives, and any combinations thereof; and at least one tribasic acid.

Surprisingly, it has been found that a solid composition comprising the first component and at least one tribasic 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 biofilm, although the invention is not limited as such.

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 include one or more tribasic acids. Therefore, the composition may include two, three, four or more tribasic acids. Typically the composition includes 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.

The tribasic acid may be in the form of granules, flakes, powders or solutions. Typically, the 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 obtained by dissolving a certain amount of tribasic acid, usually in the form of a powder, in a certain volume of water and/or 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.

Typically, 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 part 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 commonly referred to as substances that kill or inhibit the growth of microorganisms. It is generally accepted in wound healing that for a substance to exhibit antimicrobial efficacy, it must exhibit a Log4 bacterial kill rate.

The term 'antimicrobial' is used herein to describe an agent or substance capable of demonstrating a kill rate of Log4 bacteria within 24 hours. Conversely, the term 'non-antimicrobial' is used herein to refer to an agent or substance that exhibits less than a Log4 rate of bacterial kill 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 first component may be in the form of fibers, granules, flakes, powder, or combinations thereof.

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

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

Alternatively, the composition may include separate portions of the first component and the tribasic acid. For example, the first component may be in the form of fibers, granules, flakes, powder, one or more sheets, or combinations thereof, and the tribasic acid may be in the form of granules, flakes, or powder, whereby the first component and the tribasic acid are located in separate portions of the composition. The individual parts can, 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. Therefore, the solid composition may include a first component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives, and combinations thereof; and at least one tribasic acid, where the tribasic acid is associated with the carrier material.

The tribasic acid may be absorbed into or applied to the carrier material. The carrier material may act as a carrier for the tribasic acid. In such embodiments, the tribasic acid must not react 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, without limitation, polymers such as cellulose, cellulose derivatives (eg, ethylcellulose, methylcellulose, etc.), cotton, alginate, rayon, polypropylene, polyethylene, or any combination of such materials. Preferably the carrier material is viscose.

Typically, the carrier material is fibrous. In some embodiments of the invention, the first component and the carrier material can be mixed together to obtain a nonwoven product. The first component and the carrier material may be attached or sewn 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 previously explained.

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

Typically, the tribasic acid, usually in powder form, dissolves in water to form an acidic solution. The acidic solution can then be mixed with the solvent. The first component is then mixed with the acid solution/solvent mixture. The solvent and optionally part of the water can be removed by evaporation, for example, to obtain a solid composition according to the present invention. Typically, the composition comprises a tribasic acid supported on the first component.

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

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

It has been observed that the preparation of a solid composition of the present invention without initial dissolution of the first component in a solvent, and/or when the first component is not dissolved in an acidic solution/solvent mixture, allows the effective preparation of a composition having a greater amount of the first component that can be delivered to the wound. When using a first component that initially dissolves in the solvent, it is preferred that the total amount of the available first component is dissolved in the presence of the solvent. Moreover, in situations where the first component dissolves upon receipt, the final composition may not be in the form of fibers, granules or powder, which limits the potential application and form of the composition. The first component is therefore insoluble.

In cases where the first component includes 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 from about 5-80%, preferably from about 20-60%, and most preferably from about 40-50%.

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

The tribasic acid may be present in an amount of about 2-75% of the first component, preferably about 10-75% of the first component, more preferably about 20-75% of the first component, and most preferably about 25-60% of the first component. the first component. Good results were observed with the tribasic acid present at about 60% of the first component. Percentage acid refers to the relative amounts of 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, the composition comprising 20% tribasic acid contains 0.2 g of tribasic acid.

It was found in the study that large amounts of tribasic acid in the composition lead to greater effectiveness against microorganisms. Therefore, the composition may include at least about 25% tribasic acid, preferably at least about 35%, more preferably at least about 50%, and most preferably at least about 60% of the first component.

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

The term 'derivative' is used herein to refer to a compound that is derived from chitosan or chitin after one or more chemical reactions or modifications. One or more chemical reactions or modifications may include the replacement of one or more amino or hydroxyl protons in chitosan or chitin; or partial deacetylation of chitin. For example, a chitin derivative may include partially deacetylated chitin, which may have a different percentage 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 in the terms 'chitosan or chitin derivatives' are reaction products of chitosan or chitin with other compounds. Such reaction products include, without limitation, carboxymethylchitosan, hydroxylbutylchitin, N-acylchitosan, O-acylchitosan, N-alkylchitosan, O-alkylchitosan, N-alkylidenechitosan, N-arylidenechitosan, O-sulfonylchitosan, sulfated chitosan or chitin, phosphorylated chitosan or chitin, nitrated chitosan or chitin, deoxyhalochitosan, alkalichitin, alkalichitosan, or metal chelates with chitosan, organic salts, etc. Chitin or chitosan derivatives, including those referred to herein, may also contain functional groups linked to them through covalent or non-covalent connections.

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

Chitosan is a solid waste derivative of shellfish processing and can be extracted from mushroom culture. It is a cationic polymer material that is insoluble in water. Chitosan is a known hemostatic agent for use in wound dressing. The term 'hemostatic agent' is used herein as referring 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 area. human or animal wounds.

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 can have different molecular weights, different degrees of deacetylation, different arrangements of β-(1-4)-linked D-glucosamine and N-acetyl-D-glucosamine monomers, different chiral forms, or they can be obtained from different species or sources. (and mushrooms), or may be processed in various ways during production. Each and all of these different variations of chitosan materials are used for use in the present invention.

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

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

The chitosan and/or the chitosan derivative may be in any suitable form such as, for example, fibers, granules, powder, flakes, sheet, pellets, and combinations of two or more of the foregoing. Typically, the chitosan and/or the 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 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 may have a viscosity greater than 150 centipoise.

The composition of the present invention may be in the form of granules, flakes, fibers, powder, non-woven textiles or cross-linked textiles. 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 nonwoven fibers or nonwoven textiles.

The composition of the present invention may be insoluble in physiological fluid. The term 'body fluid' is used herein to refer to any fluid excreted by the human or animal body, including blood and various components of blood, saliva, sweat, urine, and the like.

The composition may be applied to a delivery mechanism.

The delivery mechanism may include polyurethane foam, polyurethane film, woven textile, superabsorbent material, medical device such as a catheter, stent, and the like. The delivery mechanism 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 that is water-swellable but not water-soluble and that is capable of absorbing liquid up to more than 2000% with a liquid retention of more than 85%. Preferably, the superabsorbent material is capable of absorbing liquid up to more than 2500% with a liquid retention of more than 90%.

The term 'water-swellable' is used herein to refer to a material that, when in contact with water or a liquid containing water, will absorb liquid and swell, but will not substantially dissolve in such liquid.

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

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

The terms "crosslink" and "crosslinked" 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 as referring to embodiments of the invention where 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, 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 a 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 between 20-75 mm, more typically between 32 and 51 mm.

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

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

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

The composition 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, wherein the fibers of the first component are at least partially coated with a tribasic acid sheath.

Typically, the composition includes a fibrous chitosan first component at least partially coated with a tribasic acid shell. Preferably, the composition includes a non-woven fibrous chitosan first component at least partially coated with citric acid. Preferably, the composition also includes a non-woven fibrous chitosan first component substantially completely coated with citric acid.

The composition of the present invention may further include a solvent acid.

The term 'solvent acid' is used herein to refer to an acid which, when applied to or associated with the first component, renders the first component more soluble in aqueous body fluids.

The composition may include one or more solvent acids. The composition may therefore include two, three, four or more solvent acids. Typically the composition includes a single solvent acid.

The solvent 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 solvent acid may preferably be a monobasic acid.

The term 'monobasic acid' (which may also be referred to herein as 'monobasic acid') 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 monobasic molecule has one substitutable 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 these.

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 obtained by dissolving some solvent acid in a volume of water and/or solvent.

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

The solvent 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, tribasic and solvent acids can be contacted with separate parts of the first component and then combined into a composition. For example, when the first component is in the form of fibers, a selection of fibers may be partially or completely coated with a tribasic acid and a separate selection of fibers may be partially or completely coated with a solvent acid.

Typically, the solvent acid is mixed with the 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 on, 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 applied to substantially the entire surface of the first component. In some embodiments, the tribasic acid may be absorbed into, or applied to, at least a portion of the first component, and the solvent acid may then be absorbed into, or applied to, at least a portion thereof, or vice versa.

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

In some embodiments, at least one tribasic acid and at least one solvent acid may be associated with the same or separate carrier materials. The composition may therefore include a first 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 solvent acid, where the tribasic acid and/or solvent acid are associated with one or separate carrier materials.

The solvent acid may be associated with the carrier material in the same manner as described herein for the tribasic acid.

The solvent acid may be applied to the first component and the tribasic acid may be applied to the carrier material, or vice versa.

The first component may include solvent acid coated fibers and the carrier material may be tribasic acid coated. The carrier material may be viscose.

The at least one solvent acid may be applied to the first component and/or carrier material by the same means as described for the tribasic acid.

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

The solvent acid is preferably provided 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%.

The at least one solvent 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.

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

It has been found in studies that a composition having a solvent acid content of about 15-30% of the first component can reduce the amount of tribasic acid required to produce the desired effect. For example, a composition having a tribasic acid content of more than 20% has been shown to give 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 solvent 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 ratio of at least one tribasic acid to at least one solvent acid is at least 1:1.

According to a further aspect of the present invention, wound dressings are provided comprising a composition as described herein.

The wound dressing may be a wound dressing that forms a gel upon contact with a liquid, such as wound exudate. In the field of gel-forming wound dressings that focus on arresting blood flow from a bleeding wound, it is particularly surprising for the composition of the present invention that the tribasic acid on non-woven wound dressings does not tend to gel and has a negative effect on water absorption and retention.

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

The composition of the present invention or wound dressing, including such a composition, may also include additional components. Such additional components include, without limitation, antimicrobial agents; pharmaceuticals; complexing agents; wetting agents such as surfactants; growth factors; cytokines; agents that absorb agents that delay 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 for tribasic and/or solvent acids.

The composition of the present invention may be mixed with other compositions useful in wound care. In some embodiments of the invention, the composition of the present invention may be mixed or mixed with one or more hemostatic agents. 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, a composition of the present invention as described herein may be mixed with a hemostatic agent such as Celox® beads, a commercially available chitosan-based hemostatic agent.

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

In accordance with a further aspect of the present invention, a composition as described herein is provided for use in the treatment of wounds.

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

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

In accordance with a further aspect of the present invention, a process is provided for preparing a solid composition comprising a first component selected from the group consisting of chitosan, chitin, chitosan derivatives, chitin derivatives, and combinations thereof; at least one tribasic acid, the method includes the steps of:

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

(b) absorption into at least one part of the first component of at least one tribasic acid.

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

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

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

(c) mixing the tribasic acid solution or the mixed acid solution with the solvent; and

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

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

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

Additional aspects of the present invention may include any or all of the characteristics described in relation to the first aspect of the present invention, if desired or if 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 test results using the MBEC assay for Pseudomonas aeruginosa ATCC 13359;

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

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

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

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

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

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

Fig. 8: is a graph showing test results 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;

Fig. 11: is a graph showing the amount of viable Staphylococcus aureus recovered from a previously formed 24 hour biofilm after 24 hours of treatment with each agent;

Fig. 12: is the amount of viable Staphylococcus aureus recovered from a preformed 72 hour biofilm 24 hours after treatment with each agent.

General method for obtaining a sample

General method 1: First component and tribasic acid

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

General method 2: First component with tribasic acid and monobasic acid

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

Exemplary Compositions

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

Example 1:

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:

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:

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:

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:

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

Example 15:

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

MBEC 1 analysis

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

The MBEC assay is a high yield screening assay used to determine the effectiveness of antimicrobial agents against biofilms from various microorganisms. The MBEC Biofilm Inoculator consists of a plastic cap with 96 pins and a matching base. There are two types of bases that can be used with the MBEC cover. One base contains 96 individual cells. Separate cells allow a variety of microorganisms to grow on the same lid pins. Another type of base is profiled through the base, which may contain only a single microorganism. Biofilms are mounted on pins under general conditions (no flow of nutrients into or out of a single cell) with gentle agitation. The resulting biofilm was transferred to a new 96 well plate to study the effectiveness of antimicrobial agents. The design of the study allows simultaneous testing of multiple biocides at multiple concentrations with repeated samples, making it an effective screening tool.

Microorganisms tested

Pseudomonas aeruginosa ATCC 13359

Staphylococcus haemolyticus

MRSA 308

Samples tested

Control: Phosphate buffer solution (PBS)

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

Sample B: Non-woven fibers of carboxymethyl cellulose with Ag (nominally 1%) (comparative sample - Aquacel Ag®)

Sample C: 100% Non-woven chitosan fibers with lactic acid to obtain a nominal composition of 25% monobasic acid (comparative sample)

Sample D: 100% Chitosan non-woven fibers with acetic acid to obtain a nominal composition of 25% monobasic acid (comparative sample)

Sample E: 100% Chitosan non-woven fibers with citric acid to obtain a nominal composition of 25% tribasic acid (example 3)

Obtaining a bacterial inoculum

A 24 hour culture of each microorganism was harvested from Trypton Soy Agar (TSA) or Brain Heart Agar (BHIA) plate and suspended in either 20 ml Trypton Soy Broth (TSB) or 20 ml Brain Heart Broth ( BHIB). The resulting bacterial suspension was diluted to obtain the initial OD590=0.10±0.03, which corresponds to a bacterial concentration of 108 CFUml ^-1 . This initial inoculum was serially diluted an additional six times to obtain a progressively lower bacterial load (ie 10 ⁷ , 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ CFUml ^-1 ). The initial bacterial concentration for each organism was typically 1×10 ⁸ ±5×10 ⁷ CFUml ^-1 .

MBEC Analysis

A 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 post caps were removed, washed briefly in phosphate buffered saline (PBS) to remove planktonic bacteria, and then placed in the agent selection plate for 24 hours. To obtain the tablet of choice for wound dressings, 1 cm ² pieces were cut using sterile scissors and placed in the designated wells of the microtiter plate. The plate of choice for the agent bead test was prepared by weighing 30 mg ± 3 mg of each bead formulation into the wells of a microtiter plate. The wound dressings and beads were then activated with 150 μl PBS. After treatment, the pin projections on the lid were washed twice in PBS, then transferred to 200 µl of neutralizer and placed in an ultrasonic water bath for 5 minutes in order to recover the remaining attached bacteria. Serial dilutions were made on the resulting reconstituted medium and drop plates were used to quantify the reconstituted bacteria. All samples were tested three times unless otherwise noted.

The result is shown in Table 1 and FIG. 1-3. The results in FIG. 1-3 refer to samples B, C and E.

It is clear from the graphs shown in Fig. 1-3 that Sample E, comprising tribasic acid coated chitosan, is effective against all three tested microorganisms. Sample C, comprising monobasic acid coated chitosan fibers, showed microorganism concentrations for all three tested microorganisms. Finally, sample B, which included 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 (2nd 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.

Microorganisms tested

Staphylococcus haemolyticus NCTC 11042

Pseudomonas aeruginosa ATCC 10434

Methicillin-resistant Staphylococcus aureus 308

Samples tested

Control: phosphate buffer solution (PBS)

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

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

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

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

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

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

Samples were obtained following the methods described previously.

Bandage samples were cut prior to use into pieces of approximately 1.5 cm ² . Phosphate buffer solution (PBS) was used as a control.

Obtaining a bacterial inoculum

A 24 hour culture of test bacteria was harvested from a trypton soy agar (TSA) plate using a sterile sponge and resuspended in 20 ml trypton soy broth (TSB). The bacterial suspension was diluted to obtain OP590=0.10±0.03, which corresponds to a bacterial concentration of 108±5×10 ⁷ CFUml ^-1 . It was further diluted in TSB and used as an inoculum for the CDC reactor containing test samples. The CDC reactor was incubated for 48 hours at 37°C, shaken at 50 rpm to stimulate biofilm growth.

Biofilm processing

After 48 hours, samples were removed from the CDC reactor and washed 3 times in sterile PBS to remove planktonic bacteria. The washed samples were then processed by placing the sample between two 1.5 cm ² dressing samples. The dressings were activated prior to testing by adding 350 μl of PBS (75% saturation) to each 1.5 cm ² piece. Control samples were immersed in 2 ml PBS for P. aeruginosa (or in the case of S. haemolyticus and MRSA in 2 ml PBS + 0.1% TSB). All samples were tested in triplicate. Microorganisms 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 clear from the graphs shown in Fig. 4-6 that samples G, H and K were effective against all three tested microorganisms. Samples G and H include chitosan fibers coated with tribasic acid and monobasic acid. Sample K includes chitosan fibers coated with a large 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 less than about 25% or less. Also, the results show that incorporating a monobasic acid with a tribasic acid can be effective in reducing the amount of tribasic acid required, eg, 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.

Microorganisms tested

Staphylococcus haemolyticus NCTC 8325

Pseudomonas aeruginosa ATCC 10434

Samples tested

Control: phosphate buffer solution (PBS)

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

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

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

Example O: 100% Chitosan non-woven fibers.

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

Sample Q: Non-woven carboxymethylcellulose with an ionic composition against biofilm formation containing silver.

Samples were obtained following the methods described previously.

Bandage samples were cut prior to use into pieces of approximately 1.5 cm ² . Phosphate buffer solution (PBS) was used as a control.

Obtaining a bacterial inoculum

A 24 hour culture of each microorganism was harvested from a trypton soy agar (TSA) plate and resuspended in 20 ml trypton soy broth (TSB). The resulting bacterial suspension was diluted to obtain the initial OP590=0.10±0.03, which corresponded to a bacterial concentration of 10 ⁸ ±5×10 ⁷ CFUml ^-1 . It 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 hours at 37° C., shaking at 50 rpm to facilitate biofilm growth.

Biofilm processing

After 24 hours and 72 hours, samples were removed from the CDC reactor and washed 3 times in sterile phosphate buffered saline (PBS) to remove planktonic bacteria. The washed samples were then processed by placing each sample between two pieces of wound dressing material. Bandages were activated before testing by adding 350 μl of PBS containing 1% TSB. Control samples were immersed in 2 ml PBS containing 1% TSB. After a treatment period of 24 hours, the samples were placed in 2 ml of neutralizer and sonicated for 15 minutes to recover the remaining attached bacteria. Serial dilutions of the resulting reconstituted medium were made and drop plates were used to quantify the reconstituted bacteria. All samples were tested in triplicate.

The results are shown in FIG. 7-10.

In FIG. 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.

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

In FIG. 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.

In FIG. 10 shows the number of viable Staphylococcus aureus recovered from preformed 72 hour biofilms after 24 hours of treatment with wound dressing samples. The control was treated with PBS+1% TSB.

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

MBEC 2 analysis

Test microorganism

Staphylococcus aureus NCTC 8325

Tested Agents

Control Phosphate buffer solution (PBS)+1% tryptone soy broth one 2.5% chitosan with 50% citric acid and 25% lactic acid 2 Chitosan with 30% citric acid and 25% lactic acid 3 Chitosan with 50% citric acid and 25% lactic acid four Chitosan with 15% citric acid and 25% lactic acid 5 Chitosan with 50% citric acid

All test agents were prepared by coating chitosan beads having a >75% deacetylation degree with specific acid(s) using a non-aqueous solvent. This gave solid chitosan salts in the form of granules. The granules were then mixed into a gel composition using deionized water with a gel containing between 5-10% chitosan salt.

MBEC Analysis

The S. aureus biofilm was grown on pin projections on the lid of the microtiter plate for 24 hours and 72 hours at 37°C. After 24 hours and 72 hours, the post caps were removed, washed briefly in sterile phosphate buffered saline (PBS) to remove planktonic bacteria, and then placed on the agent selection plate for 24 hours. To obtain a plate of choice, a certain amount of each gel composition was placed in the wells of the microtiter plate. The pin projections on the lid of the positive and negative controls were placed in PBS+1% TSB. After processing, the projections of the pins on the lid were washed twice in PBS and then placed in a neutralizer. The plates were treated with ultrasound. Serial dilutions were made on the resulting reconstituted medium and drop plates were used to quantify the reconstituted bacteria. All samples were tested in triplicate.

Twenty-Four Hour Biofilm

All agents tested successfully reduced viable bacteria recovered from pre-formed 24-hour S. aureus biofilms after 24-hour treatment at 37°C, as shown in FIG. 11. This is represented by a 5.84 ± 0.53 log reduction in viable bacteria compared to the untreated control.

Seventy two hour biofilm

All agents tested successfully reduced viable bacteria recovered from preformed 72-hour S. aureus biofilms after 24 hours treatment at 37°C, as shown in FIG. 12. For each agent, this is represented by a 5.52±0.22 log reduction in viable bacteria compared to the untreated control.

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

Claims (24)

1. A solid composition for destroying and killing bacteria in a biofilm and preventing the formation of a biofilm, comprising a first component, which is chitosan, and a tribasic acid, which is citric acid, and which is present in an amount of at least 10 wt.% of the amount of the first component, wherein citric acid is applied to at least a portion of the first component and/or absorbed into at least a portion of the first component. 2. The composition of claim. 1, where the composition is insoluble in physiological fluid. 3. A composition according to any one of the preceding claims, wherein the first component is not antimicrobial. 4. The composition according to any one of the preceding claims, wherein the weight ratio of the first component to at least one tribasic acid is at least 2:1. 5. A composition according to any one of the preceding claims, wherein the chitosan has a deacetylation degree of at least 70%. 6. The composition according to claim 5, wherein the first component has a viscosity of more than 150 centipoise in a 1% acetic acid solution. 7. A composition according to any one of the preceding claims, wherein the first component is in the form of fibers, granules, flakes, powder, or any combination thereof. 8. Composition according to claim 7, wherein the first component is in the form of fibers. 9. Composition according to claim 8, wherein the fibers are non-woven. 10. A composition according to any one of the preceding claims, wherein citric acid is present in an amount of about 25-60% by weight of the first component. 11. A composition according to any one of the preceding claims, wherein the composition is in the form of granules, flakes, fibers, powder, non-woven textiles or knitted textiles. 12. The composition of claim. 11, where the composition is in the form of non-woven fibers. 13. Composition according to any one of the preceding claims, wherein citric acid is applied to the carrier material. 14. Composition according to claim 13, where the carrier material is viscose. 15. Composition according to any of the preceding paragraphs, in addition, including an additional component selected from the group consisting of antimicrobial agents; pharmaceuticals; chelating agents; moisturizers; growth factors; cytokines; agents that absorb agents that delay healing such as MMPs (matrix metalloproteinases) and elastase; calcium; vitamin K; fibrinogen; thrombin; factor VII; factor VIII; clay; oxidized regenerated cellulose; gelatin; and collagen. 16. The composition of claim 15, 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 of them. 17. A wound dressing comprising a composition as claimed in any preceding paragraph. 18. Composition according to any one of paragraphs. 1-16 for use as a therapeutic agent. 19. The composition according to any one of paragraphs. 1-16 for use in the treatment of wounds. 20. The composition according to any one of paragraphs. 1-16 for use in disrupting and killing bacteria in a biofilm. 21. The composition according to any one of paragraphs. 1-16 for use in the prevention of biofilm formation. 22. A method for producing a composition for destroying and killing bacteria in a biofilm and preventing the formation of a biofilm, comprising a first component, which is chitosan and a tribasic acid, which is citric acid, which is present in an amount of at least 10 wt.% of the amount of the first component, the method , includes the stages: a. coating at least one part of the first component with citric acid; and/or b. absorption in at least a portion of the first citric acid component.

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