Field of the Invention

The invention concerns a wound dressing adapted for the electrochemical production of hydrogen peroxide; the use of said wound dressing to treat a wound and a method for treating a wound involving the use of said dressing.

Background of the Invention

The efficient healing of wounds is a desirable objective to maintain the quality of life and prevent morbidity. Acute and chronic wounds heal at different rates A chronic wound is a wound that does not heal in an orderly set of stages and in a predictable amount of time; wounds that do not heal within three months are often considered chronic. Moreover, chronic wounds often seem to be detained in one or more of the phases of wound healing. For example, chronic wounds often remain in the inflammatory stage for too long. Additionally, chronic wounds appear to have lost the balance between the production and degradation of molecules such as collagen and so degradation plays too large a role. In contrast, acute wounds appear to have maintained this balance and wound healing proceeds in a predictable fashion.

To make matters worse, chronic wounds (e.g. diabetic ulcers and bed sores) are typically infected and frequently exhibit antibiotic resistance, which makes them costly to treat. Frustratingly, chronic wounds may never heal or may take years to do so. Further, these wounds can cause patients severe emotional and physical stress and often lead to amputation. Chronic wounds therefore are an enormous, and growing, economic and health problem.

Wound healing is aided by treatment with a suitable therapeutic such as a reactive oxygen species (ROS), primarily hydrogen peroxide. ROS or hydrogen peroxide play a pivotal role in several aspects of wound healing, including regulating the formation of new blood vessels (angiogenesis), destroying pathogens (phagocytosis), and recruiting lymphoid ceils to the wound site for tissue repair and remodeling.

The use of ROS has been proposed as a promising means for improving chronic wound healing, the goal of which is to steer the wound from, for example, the stalled inflammatory state back onto the normal (acute) wound healing pathway. The role ROS play in the orchestration of the wound healing response together with the potential manipulation of ROS as a therapeutic avenue and infection control is reviewed in Ref 1.

However, it is important to ensure that a wound is exposed to the appropriate level of hydrogen peroxide i.e. one that ensures improved healing given the stage and condition of the wound. Accordingly, the invention concerns the efficient electrochemical production of an effective amount of hydrogen peroxide in a wound dressing.

The electrochemical, conductive polymer-mediated, oxygen reduction pathway to hydrogen peroxide and/or water is known in particular, it has been established that, for eiectropolymerized FEDOT cathode materials subjected to an applied voltage of about -0.45 V, approximately half of the current produces hydrogen peroxide and the balance produces water. However, at more negative voltages, e.g. -0.65 V, hydrogen peroxide will also be reduced to water, thereby reducing the overall amount of hydrogen peroxide produced by the electrochemical oxygen reduction reaction (Ref. 2).

Statements of the Invention

According to a first aspect, there is provided a wound dressing adapted for the electrochemical production of hydrogen peroxide comprising:

a substrate for applying to a wound having attached thereto or associated therewith a working electrode, a counter electrode and a power source for creating a voltage across said electrodes;

wherein said working electrode is arranged on the wound-facing side of said substrate and comprises a conductive conjugated polymer.

Reference herein to a conductive conjugated polymer is a polymer or macromolecule characterized by a backbone chain of alternating double- and single-bonds. Typically, their overlapping p-orbitals create a system of delocalised rr-e!eetrons, which result in useful conductive properties.

As mentioned above, it is important to ensure that a wound is exposed to an appropriate amount of hydrogen peroxide. The redox midpoint potential and the overall conductivity of the conductive conjugated polymer affect hydrogen peroxide production. It is, therefore, preferred that the conductive conjugated polymer has a redox midpoint potential, measured vs the standard hydrogen electrode (‘SHE’), between ÷0.8 V and -0.6 V to ensure that the reduced form of the polymer is sufficiently reactive towards O2. Likewise, it is preferred that the conductive conjugated polymer has a conductivity between 0.1-3000 S/cm.

Accordingly, in a preferred embodiment of the invention said conductive conjugated polymer

5 has a redox midpoint potential, measured vs SHE, between +0.8 V and -0.6 V, more preferably between ÷0.6 V and -0.6 V, still more preferably between ÷0.4 V and -0.5 V, and most preferably between ÷G 3 V and -0.4 V.

In a further preferred embodiment of the invention, said conductive conjugated polymer has0 a conductivity between 0.1-3000 S/cm, most typically 0.3 S/cm or 0.3-1000 S/cm. Yet more preferably still, it has a conductivity within one of the following ranges 30-300 S/cm, 10 ² - 10 ³ S/cm, 10-10 ³ S/cm.

In a yet further preferred embodiment of the invention said conductive conjugated polymer is5 biocompatible and, most ideally, is selected from substituted or preferably unsubstituted polymers from the group comprising: polythiophene; poly(3,4-ethyienedioxythiophene) (‘FEDOT’); (poly(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (‘PEDOT:PSS’); poly(3-alkylthiophene) such as poly(3-hexylthiophene); polyaniline; polypyrrole; polypyridine; poiyquinoline; polynapthyridine; po!ycarbazoie; poiyindole; polyazepine; po!yfluorene;0 polyphenylene; polypyrene; polyazulene; polynapthaiene; and polyacetylene. More preferably, the polymer is selected from the group consisting of PEDOT, PEDOT:PSS, polythiophene, polypyrrole, polypyridine, poiyquinoline, polynapthyridine and polyacetylene. Still more preferably, the polymer is selected from PEDOT and PEDOT: PSS. Most preferably, the polymer is PEDOT: PSS.

b

In a preferred embodiment said conductive conjugated polymer is doped to reduce the potential required for H2O2 reduction to H2O using, for example but not limited to, Meldola blue (MB); nickel, iron or other transition metal ion-based complexes such as the cobalt/cb!orin complex disclosed in Ref. 3; and conventional mediator compounds having a0 redox midpoint potential, measured vs SHE, between -0.003 V and -0.3V. Such suitable mediator compounds are disclosed in Ref. 4, and include 5-hydroxy- 1 ,4-napthoquinone; tetramethyl-p-benzoquinone; indigo-tetrasulfonate; 1 ,4-dihyrdroxnapthoquinone; pyocyanine; 2,5-dihydroxy-p-benzoquinone; indigo-sulfonate; 2-hydroxy-1 ,4-napthoquinone; 2-amino-1 ,4- napthoquinone; anthraquinone-1 ,5-disulfonate; anthraquinone-2,6-disulfonate;5 anthraquinone-2-suifonate; and 3,7-diamino-5-phenyl phenazinium chloride (phenosafranine). Advantageously, MB is immobilised on an electrode, preferably a FEDOT: PSS electrode, through electrostatic interactions. The use of a doping agent enables the circuitry to be used for hydrogen peroxide reduction and sensing as it enables less negative potentials to be used. The use of MB doped electrodes for this purpose is disclosed in Ref. 5

5

Most preferably said power source is adapted to create a voltage of about -0.45V (using a Ag/AgC! reference) to generate an effective amount of hydrogen peroxide. Such a voltage is sufficient to reduce O2 to the H2O2 (see Ref. 8), even after taking into consideration the need for an overpotential, the size of which will be material dependent to a small extent. At or0 around voltages of -0.45V, it is believed that about half of the current is producing hydrogen peroxide and half produces water (Ref. 2). At more negative potentials than -0.45V, the proportion producing water may increase. Therefore, the power source is preferably adapted to create a voltage of about -0.45V between the electrodes for optimum hydrogen peroxide production.

5

In a preferred embodiment, said power source generates a voltage that produces an effective amount of hydrogen peroxide between about 10 mM to about 500 mM, more preferably about 15 mM to about 300 mM, most preferably about 20 mM to 100 mM. The amount of hydrogen peroxide generated will also depend on other factors, including0 electrode surface area, volume of the wound being treated and the time for which voltage is applied.

In a preferred embodiment said wound dressing comprises a backing, such as a conventional plastic strip, with one or more portions for attaching said dressing to skin,b typically in the form of an adhesive coating. More preferably still, said dressing comprises a power source, such as a thin film, polymer or button battery, which is, ideally, attached to the backing.

The dressing, in use, has a wound-facing portion and it comprises a working electrode, for0 example a carbon film or indeed any other conventional electrode, in electrical communication with said conductive conjugated polymer.

Ideally said conductive conjugated polymer is deposited on the working electrode or provided as an electrically coupled fibre mat. Where the conductive conjugated polymer is5 deposited on said working electrode it is, preferably, deposited by electrospinning which, advantageously, provides a large electrode surface area. In a further preferred embodiment, the wound-facing portion of the backing further comprises a counter electrode, for example, a carbon or metallic film or indeed any other conventional electrode.

5

In operation, the power source is preferably electrically connected to the working and counter electrodes by a switch. A simple voltage regulator circuit may also be included. Thus ‘activation’ of the dressing is undertaken, following its application to a wound, by the use of said switch. In an alternative embodiment the dressing does not include said switch and, in0 this embodiment,‘activation’ of the dressing is achieved by completing the circuit as a result of electrode wetting by contact with fluid, preferably from the dressed wound.

In a further preferred embodiment, a current control device is included. For example, the power source and electrodes may be electrically connected to a constant current diode to5 provide a constant current between 5 mA and 500mA, preferably between 10 mA and 100 mA, typically about 50 mA

In yet a further preferred embodiment of the invention said wound dressing also comprises a third, reference, electrode.

0

According to an alternative aspect of the invention there is provided use of the aforementioned wound dressing to treat a wound.

According to yet another alternative aspect of the invention there is provided theb aforementioned wound dressing for use in the treatment of a wound.

According to yet another alternative aspect of the invention there is provided a method for treating a wound comprising the application of the aforementioned wound dressing to said wound.

0

Ideally, the wound is a chronic wound, although it may be an acute wound typically in its early stage(s) of healing.

In the claims which follow and in the preceding description of the invention, except where the5 context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as“comprises” or“comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith.

Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

An embodiment of the present invention will now be described by way of example only with reference to the following wherein:

Figure 1 is a schematic representation of a wound dressing in accordance with the invention;

Figure 2 is a schematic of a conventional opamp potentiostat circuit suitable for use in the dressing of the invention;

Figure 3 is a representation of the chemical structure of Meldo!a blue; Figure 4 shows electropolymerization of 50mM 3,4-ethylenedioxythiophene (FEDOT) in water: ethanol (60:40) containing 0.1 M sodium p-toluenesulfonate at pH 2 on a glassy carbon electrode (9 6 mm2). The build-up of the polymer layer is apparent from the widening scans.

Figure 5 shows a voitammetric scans of the electropoiymerised PEDOT in the reductive direction (5mV / s) in 0.1 KOH for stirred solutions under aerobic and anaerobic conditions versus Ag/AgCI reference electrode;

Figure 6 shows hydrogen peroxide production quantification of a wound dressing incorporating the circuitry of the invention. A hydrogen peroxide production of 26nmoi H2O2 /min/cm ² was achieved.

Conditions: H2O2 produced 64 pmol/L, PEDOT area 1cm ² Vol 4ml, Electrolysis time 10 minutes, Electrolyte PBS, pH 7.4 and Potential for H2O2 production -Q.45eV.

Methods and Examples

Referring firstly to figure 1 , there is shown a wound dressing in accordance with the invention. It comprises a backing material 1 , typically in the form of a flexible plastics polymer of a conventional nature. Non-limiting examples of suitable flexible plastics polymers include polyamides, polyurethanes, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polyethylene napthalate (‘PEN’), poly(ethylene-vinyl acetate) and polyethylene terephthalate (‘PET’). Preferably, the backing material is formed from a polyurethane polymer. Attached to the working side thereof or wound-facing side thereof is a working electrode 2, typically in the form of a carbon film, although other conventional forms of working electrodes may be considered such as a thin metal film, metallic mesh, conductive hydrogel, or any other suitable conductive material.

The working electrode is electrically coupled to a biocompatible conductive conjugated polymer 3. This arrangement can be achieved by depositing the conductive conjugated polymer on the working electrode in any conventional manner. For example, the conductive conjugated polymer may be in simple electrical communication with said working electrode e.g. by contacting same. Alternatively, the polymer may be deposited on the working electrode by electrospinning.

A counter electrode 4 is also provided. The counter electrode 4 is of a conventional nature and, like working electrode 2, is typically in the form of a carbon film, although other conventional forms of counter electrodes may be considered such as a thin metal film, metallic mesh, conductive hydrogel, or any other suitable conductive material. Connecting the two said electrodes is a power source 5 of a suitable nature, such as a battery, including an activating member or switch for activation of said power source.

Although not shown, a simple voltage regulator circuit may be included to apply the appropriate potential to the working electrode. Alternatively, a constant current device may be included to provide a constant current at the working electrode during operation. The current requirement at the working electrode during operation is typically in the region of 5 mA to 0.5 mA, and is preferably about 50mA.

The oxygen reduction reaction at a conductive conjugated polymer working electrode, such as FEDOT, is potential dependent. At -0 45V (using a Ag/AgC! reference) it is expected that about half of the current produces hydrogen peroxide and half produces wafer (Ref. 2). At more negative potentials, depending on how the conductive conjugated polymer, such as FEDOT, is prepared, the proportion producing water may increase. Therefore, for optimum production of hydrogen peroxide (reaction (a) figure 1), the voltage applied between the electrodes is selected for optimum hydrogen peroxide production, which is likely to be close to -0.45V using a Ag/AgCI reference.

At potentials below -0.45V, for example -0.65V versus Ag/AgC!, hydrogen peroxide is further reduced to water, as shown in figure's , reaction (b). Therefore, wound levels of hydrogen peroxide may be lowered by selecting a lower voltage potential.

With only two electrodes, the accurate control of the potential at the working electrode may be problematic because variability in the wound tissue conductivity or passivation/fouling of the counter electrode will affect the actual value at the working electrode. Better control can be achieved using three electrodes: including a working, counter and reference electrode but at the expense of added circuitry and power consumption.

For this embodiment, the reference electrode is positioned near or close to the working electrode, e.g. in a preferred embodiment, in a space in the centre of the working electrode. A thin silver film of a few mm ² area, electrochemically coated with AgCi, suffices as the reference electrode passes essentially no current.

A simple control circuit approximately functionally equivalent to a potentiostat would is also required in this embodiment. A conventional schematic opamp potentiostat circuit that would be suitable for use in this embodiment is shown in Fig.2, although a simpler transistor circuit could be used.

For hydrogen peroxide reduction and sensing (figurel reaction (b)) it is advantageous to

5 lower the overpotential as much as possible, so that less negative potentials are required and greater selectivity of measurement over, for example, oxygen reduction to water, is obtained. This can be achieved by modifying a conductive conjugated polymer, such as FEDOT, electrode with a doping agent, such as Meidola Blue (see figure 3) to lower the overpotential. This means that detection of hydrogen peroxide in the range of 5 to 120 mM,0 which encompasses the physiologically relevant range, is possible.

Electropolymerisation

Figure 4 shows eiectropolymerization of 50mM 3,4-ethylenedioxythiophene (EDOT) in water: ethanol (60:40) containing 0.1 M sodium p-toluenesulfonate at pH 2 on a glassy carbon5 electrode (9.6 mm ² ). The build-up of the polymer layer is apparent from the widening scans.

A polymer layer with a thickness of about 450 nm is suitable, although substantially thicker layers (e.g. up to 10pm, preferably from 1 to 10 pm) may be advantageous for reasons of robustness. 0 Reductive wave

Figure 5 shows voltammetric scans of the e!ectropolymerised FEDOT in the reductive direction (5m V / s) in 0.1 KOH for stirred solutions under aerobic and anaerobic conditions versus Ag/AgCi reference electrode. The oxygen reduction reaction at -0.45V (using a Ag/AgC! reference) of a conductive conjugated polymer working electrode means that aboutb half of the current produces hydrogen peroxide and half produces water. At more negative potentials, for example -0.65V versus Ag/AgCI, the proportion producing water may increase.

Hydrogen peroxide

0 Based on the results of Figure 5, and assuming that 50% of the additional aerobic current produces hydrogen peroxide, a 1cm ² electrode held at -0.45V versus Ag/AgCi in a total volume of 3 ml_ shows an increase in hydrogen peroxide concentration of about 11 pM per minute. For comparison, the physiologically relevant concentration range of hydrogen peroxide for wound healing appears to be from about 10 pM to about 100 pM (Ref. 1).

5 Taking another literature source, broadly speaking, it is suggested that 10-500mM H2O2 is beneficial for wound healing (Ref. 1) so given a wound of depth 5mm with a bandage of dimension 2cm x 2cm, a rate of generation of H2O2 of IOmM/min or above would be required to be effective within minutes. So, for this particular type of wound/dressing arrangement,

5 amount of H2O2 that needs to be produced is preferably around or above 5nmoi/min/cm ² (of electrode).

Figure 6 shows that wound dressing incorporating the circuitry of the invention, produces hydrogen peroxide in an amount of 26nmol H2O2 /min/cm ² , i.e. at an appropriate level above0 the minimum amount of 5nmol H2O2 /min/cm ² suggested above. This level of H2G2 production is sufficient to enable the design of wound dressings incorporating such electrodes into dressings that are suitable for delivering H2O2 to wounds of varying sizes and/or for which the H202 delivery rate can be tuned to a desired level by control of the applied voltage.

5

As shown above, it is possible to provide a wound dressing that eiectrochemicai!y produces hydrogen peroxide in an effective therapeutic amount for treating a wound.

References

0 1. Dunnill, C., Patton, T., Brennan, J., Barrett, J., Dryden, M., Cooke, J., Leaper, D. and

Georgopou!os, N. T. (2015), Reactive oxygen species (ROS) and wound healing: the functional role of ROS and emerging ROS-modu!ating technologies for augmentation of the healing process int Wound J, 14: 89-96. doi:10.111 1/iwj.12557.

2. Kerr, R , Pozo-Gonza!o, C., Forsyth, M. and Winther-Jensen, B. (2013), Influence of the5 Polymerization Method on the Oxygen Reduction Reaction Pathway on FEDOT. ECS

Electrochemistry Letters , 2 (3) F29-F31.

3. Mase, K., Ohkubo, K. and Fukuzumi, S. (2013), Efficient Two-Electron Reduction of Dioxygen to Hydrogen Peroxide with One-Electron Reductants with a Small Overpotential Catalyzed by a Cobalt Chlorin Complex. J. Am. Chem. Soc. 135. pp 2800-2808.

0 4. Fultz, M. L, and Durst, R. A. (1982), Mediator Compounds for the Electrochemical Study of

Biological Redox Systems: A compilation. Analytics Chemica Acta. 140. pp 1-18.

5. Siao, H. L., Chen, S. M., and Lin, K. C. (2011), Electrochemical Study of PEDOT-PSS-MDB- Modified Electrode and its Eiectrocataiytic Sensing of Hydrogen Peroxide. J Solid State Electrochem. 15. pp 1121-1128.

b 6. Wood, P M (1988), The Potential Diagram for Oxygen at pH 7. Biochem. J. 253. pp 287-289.