Dressing

Abstract

The present invention provides a wound dressing comprising: (a) a non-wound- facing porous membrane layer; and (b) a composite hydrogel layer comprising: (i) an anionic or cationic functional group-containing polymer, and (ii) at least one active pharmaceutical ingredient (API), wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer. Methods of manufacturing a composite hydrogel, methods of displacing an API from a wound dressing using an ion source, and methods of treating a wound are also provided.

Description

[0001] Dressing

[0002] Field of Invention

[0003] The present invention relates to wound dressings comprising a composite hydrogel containing at least one active pharmaceutical ingredient that can be selectively released from the hydrogel for delivery to a patient's wound.

[0004] Background to the Invention

[0005] Microbial infection is one of the principal aetiological factors in a wound becoming chronic (not healing within 6 weeks). The cost of caring for patients with a chronic wound is enormous: it is estimated to cost the UK National Health Service around GBP 3.2 billion / year to care for the 1.3 million chronic wound patients in the UK (2012 / 13 data).

[0006] It is estimated that the great majority of wound bacteria will be present as microbial communities referred to as biofilms. Biofilms are significantly harder to treat than their free-living counter parts and increase the risk of wound chronicity. Bacteria in the biofilm state are hard to remove by conventional antisepsis, often requiring 10-1000 times the minimum inhibitory concentration (MIC). Moreover, bacteria can secrete enzymes such as protease, hyaluronidase and elastase which break down dermal tissue providing a nutrient supply for the bacteria and deepening the wound; urease which raises local pH promoting further bacterial growth; and cytotoxins which cause direct cell damage.

[0007] Typically, chronic wounds are cared for by regular tissue debridement, deep cleaning, topical antiseptics and, if necessary, systemic antibiotics. However, debriding a wound also removes some healthy tissue, ultimately slows healing and risks scarring. Debridement is also often painful for the patient. In addition, the multicellular nature of biofilms can lead to antibiotic resistance, making the wound more difficult to treat. Therefore, there is a need to develop wound treatments which are more effective in treating chronic wounds and / or which allow wounds to heal without infection and scarring.

[0008] Summary of the Invention

[0009] According to a first aspect of the invention, there is provided a wound dressing comprising a non-wound-facing porous membrane layer, and a composite hydrogel layer comprising an anionic or cationic functional group-containing polymer, and at least one active pharmaceutical ingredient (API), wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer.

[0010] The wound dressing of the invention is easy to manufacture, may be kept in place on a wound for up to about 14 days, provides a moist environment to encourage wound healing and is designed to assist with exudate management.

[0011] A second aspect of the invention provides a method of manufacturing a composite hydrogel suitable for use in the wound dressing of the first aspect of the invention, the method comprising the steps of:

[0012] (i) providing anionic or cationic functional group-containing polymer particles with an average particle size of about 45 pm to about 100 pm;

[0013] (ii) mixing the anionic or cationic functional group-containing polymer particles with a natural or synthetic polymer;

[0014] (iii) adding a solution comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer particles; and

[0015] (iv) crosslinking the anionic or cationic functional group-containing polymer particles with the natural or synthetic polymer to form a hydrogel.

[0016] A third aspect of the invention provides a method of manufacturing a composite hydrogel suitable for use in the wound dressing of the first aspect of the invention, the method comprising the steps of:

[0017] (i) providing anionic or cationic functional group-containing polymer particles with an average particle size of about 45 pm to about 100 pm; (ii) mixing the anionic or cationic functional group-containing polymer particles with polyvinyl alcohol (PVA);

[0018] (iii) adding a solution comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer particles;

[0019] (iv) heating the resulting mixture at about 70 °C to about 120 °C for about 30 minutes to about 2 hours to form a gel;

[0020] (v) cooling the gel to below about 0 °C for at least about 2 hours or until the gel is completely frozen;

[0021] (vi) crosslinking the anionic or cationic functional group-containing polymer particles with the natural or synthetic polymer by freeze-thawing to form a hydrogel. Alternatively, the method may comprise steps (i), (ii), (iv), (v) and (vi) above and the API is loaded after step (vi) by soaking the hydrogel in a solution comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer particles.

[0022] The methods of manufacture described herein provide cost-effective ways to manufacture uniform, homogenous, thin composite hydrogels for use in wound dressings. The improved methods of manufacture leads to uniform API loading and distribution within the composite hydrogel, as well as improved API retention and delivery to the wound.

[0023] A fourth aspect of the invention provides a method of displacing an API from a wound dressing of the first aspect of the invention, the method comprising a step of treating the wound dressing with an ion source comprising anions, cations, or a combination thereof.

[0024] The method of displacement provided herein can be repeated to release API multiple times from the same wound dressing leading to a sustained, on-demand release of API over the period the wound dressing is used, for example about 2 weeks.

[0025] A fifth aspect of the invention provides the wound dressing of the first aspect of the invention for use in treating a wound. A sixth aspect of the invention provides a method of treating a wound comprising the steps of (i) applying the wound dressing of the first aspect of the invention to a wound, and (ii) displacing the at least one API by the method of the fourth aspect of the invention.

[0026] Description

[0027] Provided herein is a wound dressing comprising a non-wound-facing porous membrane layer that is waterproof and gas permeable so allows for consistent plasma delivery and also supports wound healing and exudate management. The wound dressing also comprises a versatile composite hydrogel layer that is easy to prepare as it does not require complex, synthetic steps, incorporates low-cost polymers and can work with a wide range of APIs.

[0028] As used herein the term "non-wound-facing porous membrane layer" means a layer of the wound dressing which does not come into contact with the wound itself. The non-wound-facing porous membrane layer may also be referred to as the top layer of the wound dressing or the external facing or the outer facing layer of the wound dressing.

[0029] As used herein the term "porous membrane" refers to a material through which gas can pass through. The size of the pores in the membrane can be tailored depending on the size of particle that are to be allowed to pass through the membrane. Preferably, the non-wound-facing porous membrane layer is waterproof so does not allow liquids to pass through it but is gas permeable, allowing gases such as air, water vapour, argon, oxygen, nitrogen or helium to pass through. The non-wound-facing porous membrane layer is preferably a vapour permeable dressing, such as a vapour permeable film dressing or a vapour permeable adhesive film dressing.

[0030] As used herein a "wound" refers to all types of tissue injury, including those resulting from burning, scalding, surgery, cutting, scratching, puncturing, bruising or medical conditions, such as autoimmune diseases or cancers. Autoimmune diseases may include psoriasis. The wound may be an open wound and may be acute or chronic. The wound may or may not be infected. The wound may be on the inside or on the outside of the body. Wound dressings as described herein can be used for the treatment of acute wounds, chronic wounds, surgical wounds, ulcers, cancers, thermal wounds, chemical, radiation-induced wounds, burns and scalds. For example, the wound dressing may be used for the treatment of burns or scalds resulting from chemical exposure, heat exposure, friction or radiation exposure. The burns may be deep or superficial. Cancers can include skin cancers and / or ulcerating cancers. The term "wound" also includes a site of infection or an area of skin or body tissue to which a pharmaceutical agent is applied.

[0031] A non-wound-facing porous membrane layer is provided to hold the composite hydrogel in the wound dressing and / or to hold the hydrogel dressing against the wound. The non-wound-facing porous membrane layer may be at least the same size, i.e., have the same surface area, as the composite hydrogel layer, or may have a greater surface area than the composite hydrogel layer. Preferably the non-wound-facing porous membrane layer is larger than the composite hydrogel layer. This allows the non-wound-facing porous membrane layer to seal the wound from surrounding skin and / or hold the wound dressing in place.

[0032] Preferably, the non-wound-facing porous membrane layer is flexible so that in use, the wound dressing can move with the body part it has been applied to. The non-wound-facing porous membrane layer may also be transparent or substantially transparent or translucent such that the wound is visible through the wound dressing. Having a transparent or substantially transparent or translucent wound dressing allows a clinician to assess how the wound is healing and if further treatment is required.

[0033] The non-wound-facing porous membrane layer of the wound dressing as described herein may comprise a hydrophobic polymer.

[0034] Examples of hydrophobic polymers include but are not limited to polytetrafluorethylene (PTFE), an acrylic polymer, an epoxy polymer, a polyolefin such as a polyethylene (PE) or a polypropylene (PP), a polystyrene (PS), a polyvinylchloride (PVC), a polydimethylsiloxane (PDMS), a polyester (PEST), a polyurethane (PU), a polyamide (PA), a polycarbonate (PC), a silicone, or combinations thereof. The hydrophobic polymer is preferably a polyurethane, such as a polyester-, polyether- or polycarbonate-based polyurethane. The polyurethane may be Mepore® film, available from Mblnlycke Ltd.

[0035] The non-wound-facing porous membrane layer may have an average pore size of about 0.01 pm to about 1 pm, preferably about 0.05 pm to about 0.8 pm, more preferably about 0.1 pm to about 0.5 pm. The average pore size of the nonwound-facing porous membrane layer may be about 0.05 pm, about 0.1 pm, about 0.15 pm, about 0.2 pm, about 0.25 pm, about 0.3 pm, about 0.35 pm, about 0.4 pm, about 0.45 pm, about 0.5 pm, about 0.55 pm, about 0.6 pm, about 0.65 pm, about 0.7 pm, about 0.75 pm or about 0.8 pm.

[0036] The non-wound-facing porous membrane layer provides a protective barrier for the wound against microorganisms such as bacteria and fungi. The average pore size is therefore preferably less than the size of the microorganism that the layer is designed to keep away from the wound. For example, if the microorganisms are about 0.025 pm in size, the average pore size of the non-wound-facing porous membrane layer should be less than about 0.025 pm. The skilled person will be able to tailor the average pore size as required.

[0037] The non-wound-facing porous membrane may further comprise an adhesive coating. The adhesive coating is preferable a polyacrylic adhesive coating. The adhesive coating can provide gentle but secure fixation of the dressing on skin. The adhesive coating is typically on the side of the non-wound-facing porous membrane that is adjacent to the composite hydrogel layer. When the nonwound-facing porous membrane is larger than the composite hydrogel layer the adhesive coating may be only on the surface of the non-wound-facing porous membrane that is not covering the hydrogel. In other words, the adhesive coating helps to secure the non-wound-facing porous membrane, and hence the wound dressing itself, to the skin but may not be present directly between the nonwound-facing porous membrane and composite hydrogel. Preferably the adhesive coating does not cause skin irritation, such as rashes or itching, and does not leave pain or discomfort upon removal.

[0038] The non-wound-facing porous membrane layer, optionally including an adhesive coating, may have a thickness of about 1 pm to about 100 pm, preferably about 5 pm to about 50 pm, more preferably about 10 pm to about 20 pm. A thinner non- wound-facing permeable membrane layer is preferred because a thinner layer allows more plasma to permeate through to the composite hydrogel layer.

[0039] The non-wound-facing porous membrane layer is preferably gas-permeable and waterproof. This allows high breathability of the wound dressing whilst providing a moist environment for wound healing. This also prevents leakage from the wound and wound maceration i.e., softening and breakdown of the skin as a result of prolonged exposure to moisture.

[0040] The non-wound-facing porous membrane layer also reduces and / or prevents drying out of the composite hydrogel and reduces and / or prevents the composite hydrogel from shrinking when plasma is applied to it through the non-woundfacing porous membrane layer. The non-wound-facing porous membrane layer also reduces and / or prevents arcing of the plasma jet which can occur when the plasma comes into contact with wet surfaces. As used herein the term "plasma arcing" refers to the effect which occurs when occurs when an electric current flows through the air from one conductive point to another. For example, from a plasma device to a patient. The heat and energy emitted by the arcing current can cause significant injury to the patient. Without being bound by theory, it is thought that plasma arcing is prevented because the non-wound-facing porous membrane layer provides a dry and homogenous layer for the plasma to interact with.

[0041] The inventors have found that providing a non-wound-facing porous membrane layer on the composite hydrogel also improves delivery of the API to the wound. Without being bound by theory, it is thought that the inclusion of a non-woundfacing porous membrane layer allows the plasma to spread out more evenly across the surface of the wound dressing, leading to a more uniform release of API from the composite hydrogel and therefore improved delivery of API to the wound.

[0042] The non-wound-facing porous membrane layer may be physically placed or layered on the composite hydrogel layer. Alternatively, the non-wound-facing porous membrane layer may be applied to the composite hydrogel layer which can be sprayed or pasted on wound. As used herein, the term "hydrogel" means a material which is not a readily flowable liquid and not a solid but a semi-solid gel which is comprised of a gel forming material, such as a hydrophilic polymer that has cross-linked or does not dissolve in water. In other words, the hydrogel may be a semi-solid substance. The hydrogel may be formed by the use of a gel forming material, such as a hydrophilic polymer which forms an interconnected, crosslinked network which can entrap, absorb and / or otherwise hold water and thereby create a gel in combination with water.

[0043] As used herein, the term "composite hydrogel" means a hydrogel material that physically or covalently incorporates particles, for example active pharmaceutical agents (API), into a crosslinked network of polymer, wherein the crosslinked network of polymer may comprise one polymer or a combination of one or more different polymers.

[0044] The composite hydrogel as described herein comprises an anionic or cationic functional group-containing polymer and at least one active pharmaceutical ingredient (API), wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer. The anionic or cationic functional group- containing polymers may be cross-linked.

[0045] The composite hydrogel may further comprise a secondary polymer which may act as a carrier gel matrix or a secondary gel matrix for the anionic or cationic functional group-containing polymer and at least one API. Examples of secondary polymers may include poly-vinyl alcohol (PVA), preferably cryo-crosslinked PVA, poly hexa-methyl methacrylate, carboxymethylcellulose, alginate and / or agarose. Preferably the polymer is PVA, such as cryo-crosslinked PVA.

[0046] Preferably the composite hydrogel has a high-water content, such as 20 wt% or more, and / or exists as a gel between the temperatures of about 20 °C and about 40 °C, wherein a gel is defined as a semi-solid substance.

[0047] As used herein, the term "anionic functional group" means any substituent with an overall negative charge. Without being bound by theory, the present inventors have determined that the anionic functional groups have an overall negative charge when the pKa of the anionic functional groups is lower than the pH of the water or solution (typically about pH 7) in which the anionic functional group- containing polymer is dispersed. When the anionic functional groups of the anionic functional group-containing polymer are negatively charged, they repel each other, so the polymer chains move apart from each other. However, due to crosslinking of the anionic functional group-containing polymers they do not dissolve in the water or solution in which they are dispersed and instead the composite hydrogel swells.

[0048] Conversely, when the pKa of the of the anionic functional groups is greater than the pH of the water or solution (typically about pH 7) in which the anionic functional group-containing polymer is dispersed, the anionic functional groups are neutralised and do not have an overall negative charge and therefore the composite hydrogel is not swollen.

[0049] As used herein, the term "pKa" refers to the negative base-10 logarithm of the acid dissociation constant (Ka) of a solution i.e., pKa = -loglOKa.

[0050] The pKa of a functional group may be measured by measuring the swelling of the hydrogel, wherein when a hydrogel is titrated with an acid, the pKa of the functional group is the point at which the hydrogel collapses into a liquid.

[0051] Examples of anionic functional groups may include, one or more of hydroxyls, carboxylates, esters, sulfonates, and phosphonates.

[0052] Examples of anionic functional group-containing polymers may be include, poly(acrylic acid) (PAA) or poly(acrylic acid) partial sodium salt-graft- poly(ethylene oxide), poly X styrene sulphonic acid, poly X maleic anhydrides, 2- Acrylamido-2-methylpropane sulfonic acid or combinations thereof.

[0053] Preferably, the anionic functional group of the anionic functional group-containing polymer comprises at least one carboxylate functional group. More preferably, the anionic functional group-containing polymer is poly(acrylic acid) (PAA).

[0054] The anionic functional group-containing polymer may comprise from about 0.5% to about 25% of the anionic functional group by weight of the total anionic functional group-containing polymer, or between 1% and 15%, or preferably between 3% and 8%. The amount of anionic functional group by weight of the total anionic functional group-containing polymer may be measured using common laboratory analytical techniques such as NMR spectroscopy, infrared spectroscopy, x-ray photoelectron spectroscopy, ultraviolet-visible spectroscopy or by titration.

[0055] As used herein, the term "cationic functional group" means any substituent with an overall positive charge. Without being bound by theory, the cationic functional groups have an overall positive charge when the pKa of the cationic functional groups is greater than the pH of the water or solution (typically about pH 7) in which the cationic functional group-containing polymer is dispersed. When the cationic functional groups of the cationic functional group-containing polymer are positively charged, they repel each other, so the polymer chains move apart from each other. However, due to cross-linking of the cationic functional group- containing polymers they do not dissolve in the water or solution in which they are dispersed, instead the composite hydrogel swells.

[0056] Conversely, when the pKa of the of the cationic functional groups is lower than the pH of the water or solution (typically about pH 7) in which the cationic functional group-containing polymer is dispersed, the cationic functional groups are neutralised and do not have an overall positive charge and therefore the composite hydrogel is not swollen.

[0057] The pKa of a functional group may be measured by measuring the swelling of the hydrogel, wherein when a hydrogel is titrated with an acid, the pKa of the functional group is the point at which the hydrogel collapses into a liquid.

[0058] Examples of cationic functional groups may include, but are not limited to, primary, secondary and tertiary amines, imines and elemental cations such as Ag+.

[0059] Examples of the cationic functional group-containing polymer may include cationic chitosan, cationic gelatin, cationic dextran, cationic cellulose, cationic cyclodextrin, Polyethyleneimine (PEI), Poly-L-lysine (PLL), Poly(amidoamine) (PAA), Poly(amino-co-ester) (PAE), Poly(2-N,N-dimethylaminoethylmethacrylate) (PDMAEMA) or combinations thereof. Preferably, the cationic functional group of the cationic functional group- containing polymer comprises at least one amino functional group.

[0060] The cationic functional group-containing polymer may comprise from about 0.5% to about 25% of the cationic functional group by weight of the total cationic functional group-containing polymer, or between 1% and 15%, or preferably between 3% and 8%. The amount of cationic functional group by weight of the total cationic functional group-containing polymer may be measured using common laboratory analytical techniques such as NMR spectroscopy, infrared spectroscopy, x-ray photoelectron spectroscopy, ultraviolet-visible spectroscopy or by titration.

[0061] The composite hydrogel layer may have a thickness of at least about 4 mm. Optionally the composite hydrogel has a thickness of about 0.5 mm to about 8 mm, preferably about 1 mm to about 4 mm.

[0062] The composite hydrogel layer comprises anionic or cationic functional group- containing polymer particles having an average particle size of about 10 pm to about 200 pm, or about 25 pm to about 150 pm, or about 45 pm to about 100 pm. The present inventors have found that using anionic or cationic functional group-containing polymer particles with an average particle size in this range leads to an improved surface area to volume ratio which allows for better plasma penetration and release of the API. As surface area to volume ratio is inversely proportional to particle size, smaller particles may be able to carry more API by unit weight of particles than larger particles.

[0063] The composite hydrogel described herein may comprise any natural or synthetic polymer suitable for use in a composite hydrogel. Suitable polymers are preferably non-toxic.

[0064] Examples of suitable natural polymers include hyaluronic acid, chitosan, heparin, alginate, fibrin and / or combinations thereof.

[0065] Examples of suitable synthetic polymers include polyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymers and / or copolymers thereof. The polymer may be crosslinked.

[0066] The wound dressing may further comprise a wound-facing porous membrane layer, which may encourage healing of the wound bed through promotion of cell migration. The wound dressing porous membrane layer may incorporate growth factors to promote wound healing. The wound-facing porous membrane layer preferably does not adhere to the wound itself and allows released API to enter the wound through it.

[0067] As used herein the term "wound-facing" means a layer of the wound dressing which is applied to the wound and makes contact directly with the wound. The wound-facing porous membrane layer may also be referred to as the bottom layer of the wound dressing or the internal facing or the inner facing layer of the wound dressing.

[0068] The composite hydrogel may be arranged / sandwiched between the non-woundfacing porous membrane layer and the wound-facing porous membrane layer. Accordingly, the non-wound-facing porous membrane layer may be a top layer, the composite hydrogel may be a middle layer and the wound-facing porous membrane layer may be a bottom layer of the wound dressing.

[0069] The wound-facing porous membrane layer may comprise a silicone polymer, a polyurethane, a polyether, a polyamide, a polyester, a polycarbonate, a polyethylene, a polyethylene oxide, a polypropylene, a non-woven polypropylene, nylon or combinations thereof. Preferably, the wound-facing porous membrane layer may comprise a polyamide, a silicone polymer or a combination thereof, for example Mepitel® film available from Mblnlycke Ltd.

[0070] The wound-facing porous membrane layer may have an average pore size of about 100 pm to about 1 pm, preferably about 50 pm to about 5 pm, more preferably about 25 pm to about 10 pm. The average pore size of the woundfacing porous membrane layer may be about 25 pm, about 24 pm, about 23 pm, about 22 pm, about 21 pm, about 20 pm, about 19 pm, about 18 pm, about 17 pm, about 16 pm, about 15 pm, about 14 pm, about 13 pm, about 12 pm, about 11 pm or about 10 pm. The average particle size of the anionic or cationic functional group-containing polymer particles is preferably greater than the average pore size of the woundfacing porous membrane layer. This prevents the anionic or cationic functional group-containing polymer particles from entering the wound.

[0071] The wound-facing porous membrane layer may have a thickness of about 5 pm to about 500 pm, preferably about 10 pm to about 300 pm, more preferably about 50 pm to about 150 pm. The thickness of the wound-facing porous membrane layer may be about 50 pm, about 75 pm, about 100 pm, about 125 pm or about 150 pm.

[0072] The size of the wound dressing is not particularly limited but may be about 60 mm by about 70 mm, or about 80 mm by about 100 mm, or about 100 mm by about 100 mm, or about 150 mm by about 150 mm, or about 200 mm by about 200 mm.

[0073] The wound dressing is preferably non-toxic and may be transparent.

[0074] As used herein, the term "active pharmaceutical agent" (API) means an ingredient in a pharmaceutical agent which is biologically or pharmacologically active.

[0075] The active pharmaceutical ingredient of the composite hydrogel as described herein may be anionic or cationic and is preferably oppositely charged to the anionic or cationic functional group containing polymer. Preferably, the API is a cationic API.

[0076] The term cationic API is used to mean an API with an overall positive charge when the pKa of the cationic API is greater than the pH of the solution containing the API.

[0077] The term anionic API is used to mean an API with an overall negative charge when the pKa of the anionic API is lower than the pH of the solution containing the API. Typically, the API is water soluble. However, APIs which are not sufficiently water soluble may be solubilised using organic solvents, emulsions, liposomes, micelles or a complexing agent. Preferably one or more complexing agents are used to increase the solubility of the API, such as cyclodextrins, for example alphacyclodextrin, beta-cyclodextrin, gamma-cyclodextrin or chemically modified cyclodextrins, for example randomly methylated beta-cyclodextrin, 2- hydroxypropyl-beta-cyclodextrin, or sulfobutylether-beta-cyclodextrin, or amino dendrimers, for example 1stand / or 2ndgeneration amino dendrimers or derivatives thereof.

[0078] The active pharmaceutical ingredient may be an antimicrobial agent. The antimicrobial agent may be a disinfectant, an antiseptic agent, antibacterial agent, an anti-biofilm agent, and / or an anti-fungal agent.

[0079] Antibacterial agents are molecules that target pathogenic bacteria by well-known methods such as interference with or inhibition of the synthesis of various bacterial cell components or pathways. Examples of suitable cationic antibacterial agents may include any antibiotic comprising functional groups with a pKa greater than the pH of the solution containing the API, for example gentamicin sulfate in water. Examples of suitable anionic antibacterial agents may include any antibiotic comprising anionic functional groups with a pKa less than the pH of the solution containing the anionic antibacterial API. pKa values of common functional groups and APIs are readily available in the literature.

[0080] Biofilms are complex structures made up of different bacterial colonies or a single type of cells in a group which proliferate and adhere to a surface. Biofilms are significantly harder to treat than their free-living counter parts and biofilm formation can increase the risk of wound chronicity. Bacteria in the biofilm state are hard to remove by conventional antisepsis, typically requiring 10-1000 times the minimal inhibitory concentration (MIC). Moreover, biofilm bacteria can secrete enzymes such as protease, hyaluronidase and elastase which break down dermal tissue providing a nutrient supply for bacteria and deepening the wound; urease which raises local pH promoting further bacterial growth; and cytotoxins which cause direct cell damage. Anti-biofilm agents are molecules that prevent the formation of biofilms by well- known methods of action such as targeting bacteria signalling molecules, targeting the extracellular polymeric substance (EPS) surrounding the biofilms, targeting the quorum sensing mechanism between bacteria in the biofilm, cleaving peptidoglycan in the bacterial cell wall, inhibiting biofilm assembly or cell division, or altering the bacterial membrane.

[0081] Examples of suitable cationic anti-biofilm agents may include any anti-biofilm agent comprising cationic functional groups with a pKa greater than the pH of the solution containing the cationic anti-biofilm agents. Examples of suitable anionic anti-biofilm agents may include any anti-biofilm agent comprising functional groups with a pKa less than 7.

[0082] Anti-fungal agents are molecules that target fungal pathogens by well-known methods such as disruption of the cell membrane, disruption of cell division, inhibition of the biosynthesis of essential sterols such as ergosterol or disruption of cell wall synthesis.

[0083] Examples of suitable cationic anti-fungal agents may include any anti-fungal agent comprising cationic functional groups with a pKa greater than the pH of the solution containing the cationic anti-fungal agents. Examples of suitable anionic anti-fungal agents may include any anti-fungal agent comprising functional groups with a pKa less than 7. Anti-fungal agents include voriconazole, flucytosine, terbinafine, clotrimazole fluconazole, toyocamycin, caspofungin, micafungin, anidulafungin, amphotericin b, chlorohexidine.

[0084] Antiseptic agents are molecules are antimicrobial substances which are nondamaging to living tissue / skin while reducing the possibility of infection, sepsis, or putrefaction. Preferred antiseptic agents include chlorhexidine, octenidine and polyhexanide.

[0085] Preferred APIs for use in the composite hydrogel include silver ions, quaternary ammonium cations (QACs), amino glycoside antibiotics, N-acyl homo-serine lactones (AHLs), anti-microbial peptides (AMPs), Polymyxin antibiotics, enzymes, Polyhexanide (also known as polyhexamethylene biguanide, PHMB), beta-lactam antibiotics and / or combinations thereof. The quaternary ammonium cation (QAC) may be cetrimide or benzethonium chloride (BTC), benzalkonium chloride (BKC) and / or the amino glycoside antibiotic may be gentamicin sulfate, amikacin or streptomycin and / or the Polymyxin antibiotic may be polymyxin B and / or the beta-lactam antibiotic may be amoxicillin. The antimicrobial peptide (AMP) may be a defensin, for example, the antimicrobial peptide may be beta-defensin.

[0086] Other suitable APIs may include anti-cancer agents, particularly anti-skin cancer agents such as adavosertib, dacarbazine or dabrafenib, anti-inflammatory agents, immunosuppressants, tissue regenerative APIs, gene therapies, antibodies, JAKs inhibitors such as ruxolitinib or tofacitinib, and / or combinations thereof. Suitable anti-cancer APIs also include chemotherapeutic agents and immunotherapies. Suitable chemotherapeutic agents may include platinum analogues (such as cisplatin, carboplatin and oxaliplatin), anthracyclines (such as doxorubicin), pyrimidine analogues (such as fluorouracil) and triazenes (such as dacarbazine and temozolomide), and / or combinations thereof. Suitable immunotherapies may include TLR-7 agonists (such as imiquimod), MEK inhibitors (such as binimetinib), antibodies (such as ipilimumab), hedgehog signalling pathway antagonists (such as vismodegib), and / or combinations thereof. Preferred anti-cancer APIs include doxorubicin, 5-fluorouracil and / or cisplatin.

[0087] The API may be present in an amount at least as high as the minimum inhibitory concentration (MIC) of the microbe (e.g., bacteria) or the minimum effective concentration (MEC) of the API or the minimum bactericidal concentration (MBC) of the API

[0088] The minimum inhibitory concentration (MIC) of the microbe (e.g., bacteria) is the lowest concentration of a substance which prevents the visible growth of a microbe (e.g., bacteria). MIC is often measured in micrograms per millilitre (pg / mL) or milligrams per litre (mg / L).

[0089] The minimum effective concentration (MEC) is the minimum concentration of an API needed in wound plasma to achieve a sufficient API concentration at receptors to produce the desired pharmacologic response. MEC is often measured in milligrams per millilitre (mg / mL) or milligrams per litre (mg / L). The MEC may be measured in wound exudate or wound tissue plasma.

[0090] The minimum bactericidal concentration (MBC) is the lowest concentration of an antibacterial agent required to kill a bacterium. MBC is often measured in colonyforming units per millilitre (CFU / mL).

[0091] The anionic or cationic functional group-containing polymer and the at least one API can be bound together to form an anionic or cationic functional group- containing polymer-API complex.

[0092] The anionic or cationic functional group-containing polymer-API complex may be bound by non-covalent interactions such as Coulombic interaction, hydrogen bonds, hydrophobic interactions, ionic bonds, dipole-dipole bonds. Preferably, the anionic or cationic functional group-containing polymer-API complex is bound by Coulombic interactions.

[0093] As used herein the term "Coulombic interaction" means the electrostatic interaction between opposite electric charges. This can also be referred to as an "ionic interaction" or an "anionic-cationic interaction".

[0094] For example, there may be a Coulombic interaction between an anionic functional group of the polymer and a cationic API at a given pH and / or a Coulombic interaction between a cationic functional group of the polymer and an anionic API at a given pH.

[0095] When the functional group of the polymer is anionic, the pKa of the anionic functional group is lower than the pKa of the cationic API.

[0096] When the functional group of the polymer is cationic, the pKa of the cationic functional group is higher than the pKa of the anionic API.

[0097] Without being bound by theory, it is believed that when the API comprises cationic functional groups, the greater the number of cationic groups i.e., functional groups with a pKa greater than 7, then the greater the binding energy of the API to the composite hydrogel matrix. Equally, when the API comprises anionic functional groups, the greater the number of anionic groups i.e., functional groups with a pKa less than 7, then the greater the binding energy of the API to the composite hydrogel matrix.

[0098] The anionic or cationic functional group-containing polymer-API complex is dispersed in the composite hydrogel.

[0099] A preferred example of the composite hydrogel system comprises sodium polyacrylate (PAA) particles, loaded with API and dispersed in a cross-linked polyvinyl alcohol (PVA) matrix. The PAA particles contain carboxylate groups which can interact with protonated primary, secondary and tertiary amine groups, often found in antimicrobials. Figure 1 shows a schematic of loading of the PAA, dispersion in PVA, and release. Without being bound by theory, it is thought that the key interactions between sodium polyacrylate and the cationic antimicrobial are Coulombic, with a pH below the pKa of the carboxylate but above that of the amine groups. This ensures the carboxylate has an anionic charge, whilst the antimicrobial remains with a net cationic charge, facilitating the Coulombic interactions.

[0100] The wound dressing may further comprise a diagnostic agent. The diagnostic agent may be incorporated into the composite hydrogel of the wound dressing. Any appropriate diagnostic agent that produces a visible signal in response to an infection or colonisation may be used. Preferably the diagnostic agent produces a visible signal when microorganisms, such as bacteria, are present at a concentration of about 104to about 106CFU / g or about 105CFU / g.

[0101] For example, the diagnostic agent may be a dye or a pH indicator or otherwise visualisable diagnostic agent that can only be visualised when an infection is present. The visualisable element may be as simple as a colour change in response to the presence of an infection, such dyes are well known. Or it might be for example a fluorescent signal that requires illumination with UV light to be visible, such a fluorescent dye.

[0102] Other possible diagnostic agents include organic or inorganic dyes such as pH sensitive dyes such as crystal blue, bromothymol blue, methyl orange, calcein and other pH or concentration sensitive fluorometric dyes. Additionally, diagnostic agents may include bacterial secreted signalling pigment molecules such as pyocyanin.

[0103] Alternatively, other chemical diagnostic agents, such as redox signalling molecules may be included.

[0104] Provided herein is a method of manufacturing a composite hydrogel suitable for use in a wound dressing as described herein, the method comprising the steps of:

[0105] (i) providing anionic or cationic functional group-containing polymer particles with an average particle size of about 45 pm and about 100 pm;

[0106] (ii) mixing the anionic or cationic functional group-containing polymer particles with a natural or synthetic polymer;

[0107] (iii) adding a solution, preferably water, comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group- containing polymer particles; and

[0108] (iv) crosslinking the anionic or cationic functional group-containing polymer particles with the natural or synthetic polymer to form a hydrogel.

[0109] Optionally, the particles of (i) and the particles of (ii) are combined at a weight ratio of about 0.5: 10 to about 2: 10. Preferably, the particles of (i) and the particles of (ii) are combined at a weight ratio of about 1: 10.

[0110] In the first step of the method, the average size of the particles is controlled. Applicants have found that controlling the average particle size of the anionic or cationic functional group-containing polymer particles allows for control of the swelling of the resulting gel that occurs. A smaller average particle size, such as a particle size of about 45 pm to about 100 pm, leads to the formation of a thin, homogenous and uniform hydrogel. Polymer particles may be ground by any suitable method in the art to provide particles of the desired size. The ground anionic or cationic functional group-containing polymer particles may also be sieved before mixing with the natural or synthetic polymer. Sieving may ensure a more uniform average particle size which may be advantageous because this can lead to a more uniform API loading and therefore more uniform API release when the wound dressing is activated. In the third step of the method, the anionic or cationic functional group-containing polymer particles are loaded with API by stirring and swelling in solution. This swelling process is controlled due to the controlled average particle size of the anionic or cationic functional group-containing polymer particles.

[0111] In the fourth step of the method, a cross-linker or initiator may be used to aid crosslinking of the polymers, depending on the anionic or cationic functional group-containing polymers and natural or synthetic polymers being used. The skilled person will be able to modify this cross-linking step to obtain the desired crosslinked hydrogel according to known methods in the art.

[0112] Provided herein is a method of manufacturing a composite hydrogel suitable for use in the wound dressing as described herein, the method comprising the steps of:

[0113] (i) providing anionic or cationic functional group-containing polymer particles with an average particle size of about 45 pm and about 100 pm;

[0114] (ii) mixing the anionic or cationic functional group-containing polymer particles with polyvinyl alcohol (PVA);

[0115] (iii) adding a solution comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer particles;

[0116] (iv) heating the resulting mixture at about 70°C to about 120°C or about 70°C to about 100°C for about 30 minutes to about 2 hours to form a gel;

[0117] (v) cooling the gel to below about 0°C, preferably to about -20°C for at least about 2 hours, preferably about 6 hours, more preferably about 12 hours;

[0118] (vi) crosslinking the anionic or cationic functional group-containing polymer particles with the natural or synthetic polymer by freeze-thawing to form a hydrogel. Preferably, at least three freeze-thawing cycles are performed, wherein one freeze-thaw cycle includes freezing the mixture below 0°C or to temperatures of between about 0°C and about -80°C or between about 0°C and about -20°C and thawing at temperatures of between about 25°C to about 45°C or between about 30°C and about 40°C.

[0119] Cycles of freezing-thawing facilitate the cryo-crosslinking of the carrier gel so that it becomes a hydrogel sheet that holds its size and shape. Without being bound by theory, it is thought that hydrogen bonds form between hydroxyl groups in the PVA polymer chain and water molecules. When the mixture is frozen, crystal growth is induced which provides a cross-linking point between polymer chains. When the mixture is thawed, the polymer chains are allowed to move freely again. As the freeze-thaw cycles are repeated, more cross-link points form and the polymer chains eventually form a matrix which traps solvent leading to the formation of a hydrogel.

[0120] Freezing makes water within polymer matrices become ice, and ice channels below 0°C do not change physical shape, so the length of time for which the hydrogel is freezing for should not affect the gel properties. Optionally the hydrogel may be frozen for about 24 hours before thawing.

[0121] Preferably the hydrogel is not thawed for too long, as the gel might be dehydrated. The hydrogel may be thawed for about 5 minutes to about 60 minutes, or about 10 minutes to about 40 minute or about 15 minutes to about 30 minutes. For example, the hydrogel may be thawed for about 20 minutes.

[0122] The methods of manufacture described herein may further comprise the step of dialysing the gel for purification, i.e., to remove any unbound API from the hydrogel matrix. The skilled person will be able to select the appropriate conditions for gel dialysis. Preferred conditions include dialysing in deionised water for about 3 to about 6 hours at a pH about 7 to about 7.5, preferably about pH 7.3. This dialysing step removes any free API molecules which are not bound to the oppositely charged anionic or cationic functional group-containing polymer particles. This is advantageous because it can stop unbound API molecules from entering the wound when the wound dressing is applied. Other hydrogel purification methods known in the art may also be used. Preferably the hydrogel is not frozen after dialysing.

[0123] The methods of manufacture described herein provide simple, low-cost and upscalable methods for obtaining composite hydrogels suitable for use in the wound dressings of the present invention. The methods avoid the need for pre-swelling of the anionic or cationic functional group-containing polymer particles in an API solution prior to hydrogel preparation. Instead, the methods of manufacture provide more control over the swelling process leading to thinner composite hydrogels, which are particularly useful in wound dressings. Suitable anionic or cationic functional group-containing polymer, natural or synthetic polymers and APIs are described above.

[0124] As described herein, the API may be displaced from the composite hydrogel layer of the wound dressing by a method which comprises the step of treating the wound dressing with an ion source comprising anions, cations, or a combination thereof. Preferably, the ion source comprises protons.

[0125] As used herein, the term "ion source" means a source which delivers ions and / or molecules that readily dissociate in aqueous media to provide ions.

[0126] Examples of suitable ion sources may include cold atmospheric plasma (CAP), plasma or dielectric barrier discharge, glow discharge, plasma activated water or plasma treated liquids, an acid and / or a mineral solution. Suitable acids include but are not limited to organic acids or mineral acids such as nitric acid hydrochloric acid, or citric acid. Suitable mineral solutions include but are not limited to calcium chloride, magnesium chloride or aluminium chloride. Preferably, the ion source is cold atmospheric plasma.

[0127] As described above, functional groups of like charge of the functional group- containing polymers repel each other, so the polymer chains move apart from each other. However, due to cross-linking of the polymer chains, they do not dissolve in the water or solution in which they are dispersed, instead the composite hydrogel swells. Conversely, when the functional groups are not charged the composite hydrogel is not swollen.

[0128] Without being bound by theory it is believed that treatment of the wound dressing with an ion source comprising anions and / or cations increases the osmotic concentration or ionic strength of the carrier gel in the composite hydrogel. This is because an increase in ions outside the composite hydrogel leads to an increase in ions inside the composite hydrogel therefore reducing the osmotic gradient. These ions may screen repulsive charges between the functional groups of like charge of the functional group-containing polymers. This charge screening leads to a de-swelling or collapse of the composite hydrogel and displacement of the API from the composite hydrogel. It is believed that this effect is stronger with divalent or trivalent cations compared to monovalent cations.

[0129] The term osmotic concentration or osmolarity is a measure of solute concentration. Osmotic concentration is typically measured in osmoles of solute per litre of solution (Osm / L).

[0130] In addition, protonation by protons in the ion source may disrupt the Coulombic interaction between an anionic species and a cationic species because the electrostatic attraction between the two charged species is lost. Accordingly, the coulombic attraction of the anionic functional-group containing polymer with the oppositely charged API, or the coulombic attraction of the anionic API with the opposite charged functional group-containing polymer is disrupted. Therefore, the API may be displaced from the composite hydrogel layer of the wound dressing.

[0131] Cold Atmospheric Plasma (CAP) is a nonthermal plasma, a type of non-equilibrium plasma in which the gas remains at relatively low temperature relative to the temperatures that are generated in thermal plasmas. Non-thermal plasmas are weakly ionised plasmas. CAP can be sustained in an inert gas for example argon, helium, nitrogen or air. When CAP is sustained in air, or when an inert gas CAP is launched into air, the result is a mix of oxygen, nitrogen and hydrogen radicals, ions, electrons, photons and ultraviolet (UV) radiation.

[0132] As used herein, the term "plasma" means plasma operated at around atmospheric pressure with the temperature of the plasma gas typically less than about 60 °C, preferably less than about 45 °C, and ideally less than about 37 °C.

[0133] The plasma can be formed using any plasma apparatus that generates a plasma stream that can be directed at a surface to be treated. The plasma apparatus may form a plasma jet, torch, needle or a dielectric barrier discharges (DBDs) such as a floating electrode configuration for treating a surface. Atmospheric pressure plasma jet devices are known in the art. Plasma jet devices can be fabricated in a multitude of electrode configurations and can be operated over a wide range of power and frequency (Hz to GHz) settings. For example, the plasma jet can be operated at a frequency of from about 10 kHz to about 30 MHz, a voltage of from about 5 kV to about 15 kV and a gas flow rate of about 0.5 litres per minute to about 10 litres per minute. The skilled person will easily be able to select suitable CAP parameters for a particular application.

[0134] The cold atmospheric plasma is excited and sustained by the application of radio frequency or microwave electrical power.

[0135] As used herein the term excited refers to when atoms and molecules in a gas become ionised, so that positive ions and free electrons result.

[0136] As used herein the term sustained refers to a continual application of radio frequency or microwave electrical power such that particles are continually ionised and the plasma is maintained.

[0137] A typical plasma jet device comprises two or more coaxially placed electrodes defining a plasma chamber there between. A plasma jet can be generated at an open end of the device by introducing a flow of gas at the other end of the device while a sufficient voltage is applied between the electrodes. A nozzle can be used at the open end to converge the plasma jet in order to obtain higher plasma densities. The plasma apparatus further comprises a power supply device for supplying electric power to the electrodes to produce plasma in the plasma chamber.

[0138] The plasma may be formed from an inert gas such as helium, argon or molecular gases such as oxygen, nitrogen, air or a combination of an inert gas with air, oxygen and / or water vapour. Optionally, the gas may also comprise an additive, such as an additive for improving the wound healing, improving the plasma characteristics or providing a sterilising effect.

[0139] As used herein, the term "inert" means a gas that does not undergo chemical reaction under given conditions.

[0140] The gas flow into the plasma chamber of the plasma apparatus is preferably controlled by a flow controller and / or an inlet valve which is arranged between a gas source and the gas inlet of the plasma apparatus. Alternatively, the plasma can be operated in ambient air with no mechanical and / or physical control over the gas flow. When the plasma apparatus is a dielectric barrier discharges (DBDs) the patient acts as the ground electrode and operating the plasma with no mechanical and / or physical control over the gas flow are preferred.

[0141] Optionally, the plasma apparatus has an ability to modulate an output to the electrodes. With this output modulation, it is possible to change the state of plasma. Note here that the output modulation refers to altering the output in characteristics to thereby change the plasma state such as pulsating the output, increasing or decreasing the magnitude of output, turning on and off the output, changing output frequency or like processing.

[0142] The plasma has a gas temperature typically below about 60°C, or preferably below about 45°C and below about 37°C when measured on the treated surface.

[0143] Without being bound by theory, it is believed that the CAP argon jet creates reactive oxygen species, primarily hydrogen peroxide which pass into the composite hydrogel matrix as a flux, but in addition there is evidence that the CAP creates oxygen and nitrogen species, for example nitric acid, nitrous acid and peroxynitric acid via interaction of the plasma with atmospheric nitrogen.

[0144] As described above, it is believed that the ion source provided by the CAP argon jet increases the osmotic concentration of the carrier gel, leading to charge screening between the functional group-containing polymers and de-swelling or collapse of the composite hydrogel. Consequently, the API is displaced / pumped out from the composite hydrogel.

[0145] Additionally, the ion source may lead to a local decrease in pH from about pH 7 to about pH 5 or less, or preferably about pH 4.5 or less which may protonate the anionic functional groups on the anionic functional-group containing polymer or the anionic API. The protonation may disrupt the coulombic attraction of functional-group containing polymer with the oppositely charged API, and reduces the interchain repulsion of the polymer chain. This decrease in repulsion creates a physical collapse of the composite hydrogel and displacement of the API. The flux of the excited plasma gas helps to carry the released API out of the composite hydrogel and into the surrounding environment, which may be water, or surrounding wound tissue.

[0146] The ion source may be applied to the composite hydrogel for a period of from about 1 minute to about 10 minutes, or from about 3 minutes to about 8 minutes, or for about 5 minutes.

[0147] The wound dressing described herein may be used in treating a wound, wherein a wound is as defined above. Treating a wound includes treating and / or preventing an infection and / or preventing colonisation by microorganisms. In other words, the wound dressing may be used prophylactically.

[0148] In one example, burn wound colonisation may be defined by concentrations of 105colony-forming units per gram (CFU / g) of microorganisms on the wound's surface. In burns and wound management one objective to prevent infection is therefore to keep the total bio-load below this threshold. APIs, such as antimicrobial agents, of the wound dressing of the present invention may be applied prophylactically to prevent exceeding this critical colonisation threshold.

[0149] The wound dressing may also be applied to a nail, such as a fingernail or toenail, before being treated with CAP to release the API, for example to treat fungal nail infections such as onychomycosis. Suitable APIs for treating fungal nail infection may include allylamine.

[0150] Wherein the wound dressing includes an API comprising antibodies or a gene therapy, the oppositely charged functional group-containing polymer may be a dendrimer such as poly(amidoamine) or part of an advanced drug delivery system. The dendrimer or advanced drug delivery system may act as a carrier or drug delivery vehicle to deliver the API to the API target site which may be on the inside or on the outside of the body.

[0151] In this scenario, the dendrimer may be bound by electrostatic charges within an anionic polymer (with a hydrogel carrier) as previously described. On application of the ion / proton source, the anionic polymer collapses releasing both dendrimer and the API, such as a gene therapy or antibody. 1

[0152] Provided herein is a method of treating a wound comprising the steps of:

[0153] (a) applying the wound dressing as described herein to a wound, and

[0154] (b) displacing the at least one API by the method as described herein, which comprises the step of treating the composite hydrogel from the non-woundfacing side with an ion source.

[0155] The wound dressing or the composite hydrogel can be applied over a wound or on a region of skin to be treated therapeutically. The wound dressing or the composite hydrogel is then treated with an ion source, preferably cold atmospheric plasma.

[0156] Accordingly, the plasma apparatus is preferably configured so that the nonthermal plasma emitted therefrom contacts the surface of the wound dressing on non-wound-facing side and / or the composite hydrogel and the plasma that passes through the wound dressing and / or the composite hydrogel displaces the API from the composite hydrogel to deliver it to the wounds or region of skin to be treated and improve the wound healing.

[0157] Provided herein is a method of delivering an API to a patient comprising the steps of:

[0158] (a) applying the composite hydrogel comprising an anionic or cationic functional group-containing polymer and at least one oppositely charged API to a patient, and

[0159] (b) displacing the at least one API by the method as described herein, which comprises the step of treating the composite hydrogel with an ion source.

[0160] Typically, the wound dressing is applied topically to a patient's wound or on a region of skin to be treated therapeutically. The wound dressing can then be treated with an ion source, preferably cold atmospheric plasma (CAP).

[0161] CAP may be applied using a multi-jet system, optionally with a distance from plasma jet orifice to wound dressing surface of from about 5 mm to about 20mm, preferably from about 5mm to about 10mm for about 5 minutes or from about 1 minute to about 10 minutes. Suitable operating parameters for the CAP system will be known to the skilled person. The patient is preferably a mammal, such as a human, a primate, bovine, ovine, equine, porcine, rodent (such as mouse or rat), feline, or canine. The patient is preferably a human and may be a paediatric or geriatric patient.

[0162] Brief Description of the Drawings

[0163] The invention will now be described in detail, by way of example only, with reference to the figures.

[0164] Figure 1 shows antimicrobial interaction with sodium polyacrylate (PAA), and Cold Atmospheric Plasma (CAP) triggered release of antimicrobial from PAA / polyvinyl alcohol (PVA) system.

[0165] Figure 2 shows the layout of a 96 well plate for testing release of drugs in a minimal inhibitory concentration (MIC) assay. This is suitable for use with all antimicrobials.

[0166] Figure 3 shows a quantification of AMC-109 release from composite hydrogel samples, using the Ninhydrin Assay. Control gels receive no CAP treatment. CAP treated gels receive 5 minutes of treatment. Control gels are shown to release a mean concentration of 118 pg / mL, whereas CAP treated gels are shown to release 388 pg / mL. Significance is assessed using an unpaired T-test, where P = 0.0379, n = 3.

[0167] Figure 4 shows a quantification of AMC-109 release following CAP treatment, where the number of dilutions that inhibit bacterial growth are used to show release of AMC-109 from the gel samples. Control gels receive no CAP treatment. CAP treated gels receive 5 minutes of treatment. Significance is assessed using an unpaired T-test, where P = 0.0013, n = 3.

[0168] Figure 5 shows a quantification of Polymyxin B release following CAP treatment, where the number of dilutions that inhibit bacterial growth are used to show release of AMC-109 from the gel samples. Control gels receive no CAP treatment. CAP treated gels receive 5 minutes of treatment. Significance is assessed using an unpaired T-test, where P = 0.006, n = 3. Figure 6 shows a quantification of Polymyxin B release over a 7-day period. The number of dilutions that inhibit bacterial growth are used to show release of AMC- 109 from the gel samples. Control gels receive no CAP treatment. CAP treated gels receive 5 minutes of treatment each day.

[0169] Figure 7a shows evidence of sustained release of PHMB into 300 pl of deionised water following daily application of 5 minute CAP to the same gel over 14 days. The cumulative drug release on day 4 (i.e., total release until day 4) was approximately 475 ug (into 1.2 ml), i.e., 395 ug / ml released in total until day 4.

[0170] Figure 7b shows evidence of sustained release of octenidene into 250 pl of deionised water following daily application of 5 minute CAP to the same gel over 7 days.

[0171] Figures 8a and 8b show enhanced release of rhodamine 6G and methylene blue dyes from composite hydrogels used with both Mepore® film and Mepitel® film layers.

[0172] Figure 9 shows release of gentamicin from Mk3 composite hydrogel without CAP; with CAP but no top film; with CAP and Mepore; with CAP and Mepitel. 5 min plasma duration.

[0173] Figures lOa-lOc show the difference in composite hydrogel shrinkage in the presence and the absence of a Mepore film.

[0174] Figure Ila shows delivery of doxorubicin from composite hydrogel after 3 and 10 minute plasma application on doxorubicin loaded gel onto cellulose. Figure 11b shows release of fluorouracil from composite hydrogel after 5 minutes plasma.

[0175] Examples

[0176] Materials and Methods

[0177] Sodium polyacrylate (PAA), polyvinyl alcohol (PVA), gentamicin sulphate salt and streptomycin sulphate were all purchased from Sigma (Poole, UK). Cetrimide was donated from Novo Nordisk Pharmatech. Tryptic soy agar (TSA), Luria-Bertani (LB) agar, Muller-Hinton (MH) agar, brain-heart infused (BHI) agar, tryptic soy broth (TSB), LB broth, MH broth were all purchased from Sigma. Whatman Polycarbonate membranes were purchased from Fischer.

[0178] From hydrogel application - PAA polymer

[0179] 'Superabsorbent' polyacrylic acid partial sodium salt-graft-poly(ethylene oxide) particles (CAS number: 9003-04-7) was obtained from Sigma-Aldrich. A further source of PAA was obtained from BASF, with the tradename SavivaTM. Saviva particles are chemically similar to the Sigma-Aldrich particles (primarily polyacrylic acid) but with a smaller particle size distribution and more spherical morphology.

[0180] Preparation of Composite Hydrogels

[0181] Sodium polyacrylate (PAA) particles were first ground in a coffee grinder and sieved using a 100 pm and a 45 pm micro sieves, to give particles between 100 pm and 45 pm. Ground and sieved sodium polyacrylate particles (0.1 g) were then thoroughly mixed with poly-vinyl alcohol (1 g) in a Schott bottle. 20 mL of antimicrobial solution, with a concentration of 1 or 2 mg / mL was prepared in deionized water (18.2 MQ-cm, pH 7.3) and added into the polymer mixture. The mixture was stirred (1200 rpm) while heating at 95 °C for 1 hour. The gel mixture was then poured into a petri dish (90 mm diameter) and frozen for 3 hours at -20 °C to facilitate the cryo-crosslinking of polyvinyl alcohol gel. Gels were defrosted for 20 minutes at 37 °C, and this freeze-thaw process was repeated a further 2 times. After 3 times of freeze-thaw process, crosslinked gels were dialysed in MilliQ water (500 mL, pH 7.3) for 4 hours, ensuring the water is changed halfway through. Gels were then rinsed and stored in a sealed container in the fridge until use. The above procedure was carried out aseptically using clean utensils to minimize contamination.

[0182] Microbiology Assays

[0183] Minimum Inhibitory Concentration Assay

[0184] Overnight cultures (1 x 109CFU / mL) were prepared: Pseudomonas aeruginosa (P. aeruginosa PAO1) Staphylococcus aureus S. aureus H560) from the Jenkins collection at the University of Bath. Candida albicans (C. albicans 2620 and 2621) from the Jenkins collection at the University of Bath. Bacteria and fungi were maintained in 15 % (v / v) glycerol at -80°C and streaked out as required onto Luria-Bertani (LB) agar, tryptic soy agar (TSA) and sabouraud dextrose agar (SDA) respectively. Overnight cultures were made by inoculating a single bacterial colony into 10 mL of broth, Muller-Hinton (MH) broth for Kirby-Bauer tests, tryptic soy broth (TSB) and BHI broth respectively for biofilm work. Overnight fungal cultures were made by inoculating a single fungi colony into 10 mL of yeast extract peptone dextrose (YPD) broth. Bacterial and fungal subcultures (1 x 106CFU / mL) were prepared by a lOOOx dilution in bacterial and fungal broths respectively. Stock solutions of antimicrobial (1.024 mg / mL) were prepared in MilliQ water (pH 7.3).

[0185] 0.2 mL MilliQ water (pH 7.3) was pipetted into well Al of a 96 well plate (see figure 2). 0.2 mL of antimicrobial stock solution was pipetted into wells Bl, Cl, and DI (to produce the MICs in triplicate.) 0.1 mL bacterial broth was pipetted into wells A2-11, B2-11, C2-11, and D2-11, and 0.2 mL bacterial broth was pipetted into A12, B12, C12, and D12. 0.1 mL of solution from each well in column 1 was then serially diluted across the plate to column 10. All wells in rows A-D, excluding column 12, were then filled with 0.1 mL bacterial subculture. Column 11 acted as the positive control, and column 12 acted as the negative control. This process was repeated for rows E-H if a second bacteria strain was to be tested. The 96 well plate was then incubated for 18 hours at 37 °C for bacteria and 33 °C for fungi. Absorbance was read at 600 nm at 18 hour time point and bacteria l / funga I growth curves were constructed. The well containing the lowest concentration that inhibited bacteria l / funga I growth was the Minimum Inhibitory Concentration (MIC).

[0186] Antimicrobial Release Quantification Assay

[0187] Overnight cultures of Staphylococcus aureus and Pseudomonas aeruginosa (1 x 109CFU / mL) were prepared as outlined. Bacterial subcultures (1 x 106CFU / mL) were prepared by a lOOOx dilution in bacterial broth. Stock solutions of antimicrobial (1.024 mg / mL) were prepared in MilliQ water (pH 7.3). Antimicrobial loaded gel discs were treated with Cold Atmospheric Plasma (CAP) as outlined or left untreated as control measures. Each disc was incubated with MilliQ water (0.5 mL per disc, pH 7.3) for 18 hours at 37 °C and 200 rpm. A 96 well plate was subsequently prepared as follows: 0.2 mL MilliQ water (pH

[0188] 7.3) was pipetted into well Al of a 96 well plate. 0.2 mL of antimicrobial stock solution was pipetted into well Bl (to provide the MICs as a control measure). 0.2 mL of each of the incubated solutions was pipetted into the remaining wells in column 1 (from 3 x CAP treated gels and 3 x control gels). 0.1 mL bacterial broth was pipetted into all wells in columns 2-11, and 0.2 mL bacterial broth was pipetted into all wells in column 12. 0.1 mL of solution from each well in column 1 was then serially diluted across the plate to column 10. All wells, excluding column 12, were then filled with 0.1 mL bacterial subculture. Column 11 acted as the positive control, and column 12 acted as the negative control. The 96 well plate was then incubated for 18 hours at 37 °C. Absorbance was read at 600 nm at 18 hour time point and bacterial growth curves were constructed. The well containing the lowest concentration that inhibited bacterial growth was the Minimum Inhibitory Concentration (MIC) in row B, and the amount of antimicrobial released from each gel could then be back calculated based on this value.

[0189] Antifungal release quantification assav

[0190] Overnight cultures (1 x 109CFU / mL) of Candida albicans were prepared as outlined. Fungal subcultures (1 x 106CFU / mL) were prepared by a lOOOx dilution in fungal yeast peptone dextrose (YPD) broth. Stock solutions of antifungal (1.024 mg / mL) were prepared in MilliQ water (pH 7.3). Antifungal loaded gel discs were treated with Cold Atmospheric Plasma (CAP) as outlined or left untreated as control measures. Each disc was incubated with MilliQ water (0.5 mL per disc, pH

[0191] 7.3) for 18 hours at 33 °C and 200 rpm.

[0192] A 96 well plate was subsequently prepared as follows: 0.2 mL MilliQ water (pH

[0193] 7.3) was pipetted into well Al of a 96 well plate. 0.2 mL of antifungal stock solution was pipetted into well Bl (to provide the MICs as a control measure). 0.2 mL of each of the incubated solutions was pipetted into the remaining wells in column 1 (from 3 x CAP treated gels and 3 x control gels). 0.1 mL fungal broth was pipetted into all wells in columns 2-11, and 0.2 mL fungal broth was pipetted into all wells in column 12. 0.1 mL of solution from each well in column 1 was then serially diluted across the plate to column 10. All wells, excluding column 12, were then filled with 0.1 mL fungal subculture. Column 11 acted as the positive control, and column 12 acted as the negative control. The 96 well plate was then incubated for 18 hours at 33 °C. Absorbance was read at 600 nm at 18 hour time point and fungal growth curves were constructed. The well containing the lowest concentration that inhibited fungal growth was the Minimum Inhibitory Concentration in row B, and the amount of antifungal released from each gel could then be back calculated based on this value.

[0194] Cold Atmosoheric Plasma Methods

[0195] Plasma Jet Treatment of Gels

[0196] Hydrogel discs (0 = 18 mm) were placed individually on the lid of a petri dish, and MilliQ water (0.05 mL) was pipetted underneath each disc. Each disc was treated with Cold Atmospheric Plasma (CAP), at a distance of 5 mm for 5 minutes, moving the gel for the duration of the treatment to ensure even application of CAP. All gels were treated with the argon multi-jet system, using the following operating parameters: Vpp = 8 kV, Frequency = 23.5 kHz, Gas flow rate = 10 SLPM, distance from plasma jet orifice to gel surface = 15 mm, treatment duration per gel = 5 minutes.

[0197] Microbioloav Methods

[0198] Growing Overnight Cultures

[0199] Appropriate growth media (10 mL) in a sterile Falcon tube is inoculated with a single bacterial colony. This was then incubated for 18 hours at 37 °C and 200 rpm. Gram-positive bacteria were grown in Tryptic Soy Broth (TSB), and Gramnegative bacteria grown in Luria Bertani Broth (LB). For fungal culture, the growth media was 10 mL of yeast extract peptone dextrose (YPD) broth, starting with a single fungal colony, incubated for 18 hours at 33 °C and 200 rpm.

[0200] Making Bacteria l / funqa I Freezer Stocks

[0201] Overnight culture of bacteria / fungi in growth media and sterile 30 % glycerol (v / v in growth media) were added to cryogenic vials in a 1: 1 ratio. The vials were vortexed, then placed in the -80 °C freezer for long-term storage.

[0202] Exemplary non-wound-facinq porous membrane layer A flexible transparent polyurethane layer from Mblnlycke Ltd. was used to hold the composite hydrogel dressing. Mblnlycke produced and marketed the layer as Mepore® film. The layer contains a polyacrylic adhesive coating.

[0203] Results and discussion

[0204] Incorporation of Antimicrobial Peptides into the Composite Hydrogel System Minimum Inhibitory Concentrations of Antimicrobial Peptides

[0205] To ensure selected antimicrobials would be appropriate for use with the hydrogel system, Minimum Inhibitory Concentration (MIC) values of Gentamicin, Polymyxin B and AMC-109 were determined. Gentamicin is aminoglycoside antibiotic used as a reference antibiotic to compare the MIC with antimicrobial peptides. Polymyxin B is an antimicrobial peptide that is already used in clinical settings. AMC-109 is a cationic antimicrobial peptide that is not yet used in healthcare. The chemical structure of AMC-109 is provided below:

[0206] As well as containing amine groups and being cationic, the antimicrobials were chosen because they have low MIC values with common bacteria species found in wounds. The bacterial strains tested were S. aureus (H560) and P. aeruginosa (PAO1), and their MICs can be seen in Table 1.

[0207] Table 1 : MICs of selected antibiotic and antimicrobial peptides for S. aureus and P. aeruginosa.

[0208] MIC values of 2-4 and 4-8 pg / mL were deemed appropriate as they are likely to be achieved on a single CAP application. Table 2 shows that whilst AMC-109 and gentamicin could potentially be suitable for treatment of both S. aureus and P. aeruginosa, Polymyxin B is only suitable for use with P. aeruginosa due to its high MIC value with S. aureus. This was key for deciding which bacteria species would be used in further experiments.

[0209] Ninhydrin Assay for Quantification of Antimicrobial Release

[0210] The Ninhydrin assay was used for quantifying drug release from the gels. The antimicrobial release was quantified by using the calibration curve produced with known concentration of drug. Samples were either treated with Cold Atmospheric Plasma (CAP) or left untreated as control measures. Each sample was incubated overnight with 500 pL of acetic acid buffer. This buffer is required for amines to be detected using the Ninhydrin reagent. Once the incubated solutions were extracted and mixed with the Ninhydrin reagent, absorbance values of each solution were measured, and used to calculate the corresponding concentrations of AMC-109. Results are shown in Figure 3 which indicate that the concentration of API released with CAP is greater than the untreated control sample.

[0211] The data shows that application of CAP treatment produces a greater release response from the hydrogel samples, compared with the untreated samples. Despite the error bars appearing relatively large, the difference in released concentration between the control and CAP treated gels is seen to be statistically significant, based on the results of the T-test.

[0212] Microbiology-based Assay for Quantification of Antimicrobial Release

[0213] This microbiology-based method was used for one-time applications of CAP for both AMC-109 gels and Polymyxin B gels. The number of dilutions required to give the minimum inhibitory concentrations for specified bacteria are shown as bar graphs in Figures 4 and 5. Comparison of Figures 4 and 5 shows that for AMC-109 hydrogel samples, 3 dilution steps are required to reach the minimum inhibitory concentration, whereas hydrogels containing Polymyxin B reach the MIC value after 8 dilution steps. This suggests that a much greater concentration of Polymyxin B is released from the gels following CAP treatment, compared with AMC-109 release.

[0214] These results highlight how the composite hydrogel system has different release profiles depending on which drug is encapsulated. As such, factors such as how many amine groups are present, or pH of the gel based on the antimicrobial used, could be tailored to control release profile.

[0215] Following the results from the initial one-time CAP application study, Polymyxin B gels were then used in a study to assess if sustained release over 7 days was possible. Hydrogel samples were treated for 5 minutes once a day for 7 days, and culture plates were prepared each day after overnight incubation. Untreated gels were also tested as a control measure.

[0216] Each sample was done in triplicate, but error bars cannot be observed as all three samples showed inhibition after the same number of dilutions, as seen in Figure 6.

[0217] Although this method does not quantify exact concentrations of antimicrobial released from the hydrogel samples, the results show that sustained release of polymyxin B over one week is possible. This is shown by the same number of dilution steps being required each day to reach the MIC, indicating that concentrations released from each treated hydrogel sample are in the same range.

[0218] Additionally, little to no leakage of polymyxin B is observed from the control gels as the MIC was not met by the solutions incubated with these gels.

[0219] Release of Polvhexa methyl biauanide (PHMB) over 14 days

[0220] Release of PHMB and octenidene was also tested, with release being quantified by Ninhydrin assay, for up to 14 days with a daily application of 5 minutes plasma jet to the loaded composite hydrogel. 31

[0221] Figures 7a and 7b show the sustained on-demand release of PHMB and Octenidine dihydrochloride over 2 weeks and 1 week respectively, with a 5 minute daily CAP application. Figure 7a shows that showed that PHMB can be delivered at concentration significantly greater than its MIC for up to 14 days.

[0222] Effect of non-wound-facing porous membrane layer

[0223] Composite hydrogels were made using Rhodamine 6G (fluorescent dye) and Methylene Blue and tested for dye release by plasma (single) jet treatment in the absence and presence of commercially available non-wound facing layers Mepore® film and Mepitel® film, available from Mblnlycke.

[0224] The results are shown in figures 8a and 8b which show that release of both of the dyes from the composite hydrogel layer was enhanced when Mepore® or Mepitel® were present.

[0225] Gentamicin release

[0226] Release of the antibiotic gentamicin following 5 minute CAP application from a composite hydrogel layer in the absence and presence of Mepore® or Mepitel® was tested.

[0227] Figure 9 shows that the release of gentamicin was enhanced in the presence of the Mepore® layer, but not in the presence of the Mepitel® layer.

[0228] Gel shrinkage following plasma treatment

[0229] Composite hydrogel shrinkage in the absence and the presence of a Mepore® layer was investigated following 3 min CAP application.

[0230] Figures lOa-c show that composite hydrogel shrinkage was greatly reduced in the presence of the Mepore® layer.

[0231] Figure 11 shows delivery of doxorubicin from composite hydrogel after 3 and 10 minute plasma application on doxorubicin loaded gel onto cellulose. Figure 11b shows release of fluorouracil from composite hydrogel after 5 minutes plasma.

[0232] Conclusion Using a gas permeable film on top of the composite hydrogel provides a number of advantages, including facilitating multiple deliveries of anti-microbial agents over time; providing a more mechanically robust dressing that resists water loss from the hydrogel and consequent shrinkage following plasma activation; a thinner wound dressing, which could potentially be made transparent to aid in visual monitoring of a wound; and reducing arcing of the plasma jet, creating a more homogenous layer for interaction with the plasma jet and enhancing drug delivery by the plasma to the wound.

Claims

Claims1. A wound dressing comprising:(a) a non-wound-facing porous membrane layer; and(b) a composite hydrogel layer comprising:(i) an anionic or cationic functional group-containing polymer, and(ii) at least one active pharmaceutical ingredient (API), wherein the API is oppositely charged to the anionic or cationic functional group- containing polymer.

2. The wound dressing of claim 1, wherein the non-wound-facing porous membrane layer comprises a hydrophobic polymer.

3. The wound dressing of any preceding claim, wherein the non-wound-facing porous membrane layer comprises polytetrafluorethylene (PTFE), an acrylic polymer, an epoxy polymer, a polyethylene, a polystyrene, a polyvinylchloride (PVC), a polydimethylsiloxane (PDMS), a polyester, a polyurethane, a silicone, or combinations thereof.

4. The wound dressing of any preceding claim, wherein the non-wound-facing porous membrane layer has an average pore size of about 0.01 pm to about 1 pm, preferably about 0.05 pm to about 0.8 pm, more preferably about 0.1 pm to about 0.5 pm.

5. The wound dressing of any preceding claim, wherein the non-wound-facing membrane comprises an adhesive coating, preferably a polyacrylic adhesive coating.

6. The wound dressing of any preceding claim, wherein the non-wound-facing porous membrane layer has a thickness of about 1 pm to about 100 pm, preferably about 5 pm to about 50 pm, more preferably about 10 pm to about 20 pm.

7. The wound dressing of any preceding claim, wherein the composite hydrogel layer has a thickness of at least 4 mm.

8. The wound dressing of any preceding claim, wherein the composite hydrogel layer comprises anionic or cationic functional group-containing polymer particles having an average particle size of about 45 pm to about 100 pm.

9. The wound dressing of claim any preceding claim, further comprising a woundfacing porous membrane layer.

10. The wound dressing of claim 9, wherein the composite hydrogel is arranged between the non-wound-facing porous membrane layer and the wound-facing porous membrane layer.

11. The wound dressing of claim 9 or 10, wherein the wound-facing porous membrane layer comprises a silicone polymer, a polyurethane, a polyether, a polyester, a polycarbonate, a polyethylene, a polyethylene oxide, a polypropylene, a non-woven polypropylene, nylon or combinations thereof.

12. The wound dressing of claims 9 to 11 wherein the wound-facing porous membrane layer has an average pore size of 100 pm to 1 pm, preferably 50 pm to 5 pm, more preferably 25 pm to 10 pm.

13. The wound dressing of claims 9 to 12, wherein the average particle size of the anionic or cationic functional group-containing polymer particles is greater than the average pore size of the wound-facing porous membrane layer.

14. The wound dressing of claims 9 to 13, wherein the wound-facing porous membrane layer has a thickness of about 5 pm to about 500 pm, preferably about 10 pm to about 300 pm, more preferably about 50 pm to about 150 pm.

15. A method of manufacturing a composite hydrogel suitable for use in the wound dressing of claims 1 to 14, the method comprising the steps of:(i) providing anionic or cationic functional group-containing polymer particles with an average particle size of about 45 pm and about 100 pm;(ii) mixing the anionic or cationic functional group-containing polymer particles with a natural or synthetic polymer;(iii) adding a solution comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer particles; and(iv) crosslinking the anionic or cationic functional group-containing polymer particles with the natural or synthetic polymer to form a hydrogel.

16. A method of manufacturing a composite hydrogel suitable for use in the wound dressing of claims 1 to 14, the method comprising the steps of:(i) providing anionic or cationic functional group-containing polymer particles with an average particle size of about 45 pm and about 100 pm;(ii) mixing the anionic or cationic functional group-containing polymer particles with polyvinyl alcohol (PVA);(iii) adding a solution comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer particles;(iv) heating the resulting mixture at about 70 °C to about 120 °C for about 30 minutes to about 2 hours to form a gel;(v) cooling the gel to below about 0 °C for at least about 2 hours;(vi) crosslinking the anionic or cationic functional group-containing polymer particles with the natural or synthetic polymer by freeze-thawing to form a hydrogel.

17. A method of manufacturing a composite hydrogel suitable for use in the wound dressing of claims 1 to 14, the method comprising the steps of:(i) providing anionic or cationic functional group-containing polymer particles with an average particle size of about 45 pm and about 100 pm;(ii) mixing the anionic or cationic functional group-containing polymer particles with polyvinyl alcohol (PVA);(iii) heating the resulting mixture at about 70 °C to about 120 °C for about 30 minutes to about 2 hours to form a gel;(iv) cooling the gel to below about 0 °C for at least about 2 hours;(v) crosslinking the anionic or cationic functional group-containing polymer particles with the natural or synthetic polymer by freeze-thawing to form a hydrogel; and(vi) soaking the hydrogel in a solution comprising at least one API, wherein the API is oppositely charged to the anionic or cationic functional group-containing polymer particles.

18. The methods of any of claims 15 to 17, further comprising the step of dialysing the gel for about 3 to about 6 hours at pH 7.3 in deionised water.

19. The wound dressing of claims 1 to 14 or the methods of claims 15 to 18, wherein the anionic functional group comprises at least one carboxylate functional group, or wherein the cationic functional group comprises at least one amino functional group.

20. The wound dressing of claims 1 to 14 or 19 or the methods of claims 15 to 18, wherein the anionic functional group-containing polymer is selected from one or more of poly(acrylic acid) (PAA) or poly(acrylic acid) partial sodium salt-graft- poly(ethylene oxide), poly X styrene sulphonic acid, poly X maleic anhydrides, 2- Acrylamido-2-methylpropane sulfonic acid or combinations thereof, or wherein the cationic functional group containing polymer is selected from one or more of polyamino acids, polyamides and polymers grafted with amine containing side chains, cationic chitosan, cationic gelatin, cationic dextran, cationic cellulose, cationic cyclodextrin, Polyethyleneimine (PEI), Poly-L-lysine (PLL), Poly(amidoamine) (PAA), Poly(amino-co-ester) (PAE), Poly(2-N,N-dimethylaminoethylmethacrylate) (PDMAEMA) or combinations thereof.

21. The wound dressing of claims 1 to 14, or 19 to 20 or the methods of claims 15 to 18, wherein the API is an antimicrobial agent or a chemotherapeutic agent.

22. The wound dressing of claim 21 or the method of claim 21, wherein the chemotherapeutic agent is selected from one or more of doxorubicin, 5- fluorouracil and cisplatin.

23. The wound dressing of claim 21, or the method of claim 21, wherein the antimicrobial agent is selected from one or more of an antibacterial agent, an antiseptic, a disinfectant, an anti-biofilm agent and an anti-fungal agent, optionally wherein the antiseptic is benzalkonium chloride (BKC), and / or the disinfectant is chlorhexidine, and / or the anti-fungal agent is voriconazole,flucytosine, terbinafine, clotrimazole, fluconazole, toyocamycin, caspofungin, micafungin, anidulafungin or amphotericin b.

24. The wound dressing of claims 1 to 14 or 19 to 21 or the method of claims 15 to 17, wherein the API is selected from one or more of silver ions, quaternary ammonium cations (QACs), amino glycoside antibiotics, N-acyl homo-serine lactones (AHLs), anti-microbial peptides (AMPs), Polymyxin antibiotics, enzymes, beta-lactam antibiotics, and / or combinations thereof.

25. The wound dressing of claim 24 or the method of claim 24, wherein the quaternary ammonium cation (QAC) is cetrimide, benzethonium chloride (BTC) or benzethonium chloride, and / or the amino glycoside antibiotic is gentamicin, amikacin or streptomycin and / or the antimicrobial peptide (AMP) is a defensin and / or the Polymyxin antibiotic is polymyxin B and / or the beta-lactam antibiotic is amoxicillin.

26. The wound dressing of claims 1 to 14 or 19 to 20 or the method of claims 15 to 20, wherein the API is selected from, an anti-cancer agent, an antiinflammatory agent, an immunosuppressant, a tissue regenerative API, a gene therapy, an antibody and / or combinations thereof.

27. The wound dressing of claims 1 to 14 or 19 to 26 or the method of claims 15 to 26, wherein the anionic or cationic functional group-containing polymer and the at least one API are bound together to form an anionic or cationic functional group-containing polymer-API complex.

28. The wound dressing of claim 27 or the method of claim 27, wherein the anionic or cationic functional group-containing polymer-API complex is bound by Coulombic interaction.

29. The wound dressing of claims 27 or 28 or the method of claims 27 or 28, wherein the anionic or cationic functional group-containing polymer-API complex is dispersed in the composite hydrogel.

30. The wound dressing of claims 1 to 14 or 19 to 29 or the method of claims 15 to 25, wherein the composite hydrogel layer comprises a natural polymer or a synthetic polymer.

31. The wound dressing of claim 30 or the method of claim 30, wherein the natural polymer is selected from one or more of hyaluronic acid, chitosan, heparin, alginate, and fibrin.

32. The wound dressing of claim 30 or the method of claim 30, wherein the synthetic polymer is selected from one or more of polyvinyl alcohol, polyethylene glycol, sodium polyacrylate, acrylate polymers and copolymers thereof.

33. The wound dressing of claims 1 to 14 or 19 to 32 or the method of claims 15 to 32, further comprising a diagnostic agent, wherein the diagnostic agent produces a visible signal in response to an infection.

34. The wound dressing of claim 33 or the method of claim 33, wherein the diagnostic agent is selected from one or more of a dye or a pH indicator.

35. A method of displacing an API from a wound dressing of any of claims 1 to 14 or 19 to 34, the method comprising a step of treating the wound dressing with an ion source comprising anions, cations, or a combination thereof.

36. The method of claim 35, wherein the ion source is selected from cold atmospheric plasma, plasma activated water, plasma or dielectric barrier discharge, glow discharge or an acid.

37. The method of claim 36, wherein the cold atmospheric plasma is a gas selected from an inert gas or a combination of an inert gas with air, oxygen and / or water vapour.

38. The method of claim 37, wherein the cold atmospheric plasma is an inert gas selected from one or more of argon, nitrogen or helium.

39. The method of any of claims 36 to 38, wherein the cold atmospheric plasma is excited and sustained by the application of radio frequency or microwave electrical power.

40. The method of any of claims 36 to 39, wherein the temperature of the cold atmospheric plasma is below about 45 °C, preferably below about 37 °C41. The method of any of claims 35 to 40, wherein the ion source is applied to the composite hydrogel for a period of from about 1 minute to about 10 minutes.

42. The wound dressing of any of claims 1 to 14 or 19 to 34, for use in treating a wound.

43. A method of treating a wound comprising the steps of:(i) applying the wound dressing of any of claims 1 to 14 or 19 to 34 to a wound, and(ii) displacing the at least one API by the method of any of claims 35-41.

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