WO2018068076A1

WO2018068076A1 - Compositions and materials for assisting in wound healing

Info

Publication number: WO2018068076A1
Application number: PCT/AU2017/000214
Authority: WO
Inventors: Anthony Richard BLENCOWE; Neethu NINAN; Nicolas Hans VOELCKER
Original assignee: University Of South Australia
Priority date: 2016-10-11
Filing date: 2017-10-11
Publication date: 2018-04-19

Abstract

A wound dressing comprising an oxygen-loaded zeolite. The oxygen-loaded zeolite releases oxygen when in use. Methods of producing an oxygen-loaded zeolite layer for the wound dressing and uses of the wound dressing are also disclosed.

Description

COMPOSITIONS AND MATERIALS FOR ASSISTING IN WOUND HEALING

PRIORITY DOCUMENT

100011 The present application claims priority from Australian Provisional Patent Application No.

20169041 15 titled "COMPOSITIONS AND MATERIALS FOR ASSISTING IN WOUND HEALING" and filed on 11 October 2016, the content of which is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

[0002 ] The present disclosure relates to the use of zeolites that bind oxygen. In a particular fonn, the present disclosure relates to the use of oxygen-loaded zeolites to assist wound healing or other biochemical processes.

BACKGROUND

[0003] The state of wound oxygenation is thought to be a key determinant of healing outcomes for wounds, particularly significant and/or chronic wounds. Providing a topical supply of oxygen to a wound (eg, ulcers, abrasions, lacerations, cuts, sores, burns, etc) is thought to promote healing of the wound. Oxygen therapy is used for inducing the growth of new skin tissue to close and heal ischemic wounds. Topical oxygen therapy calls for applying oxygen directly to an open wound. The oxygen is thought to dissolve in tissue fluids and improve the oxygen content of the intercellular fluids. Injuries and disorders which may be treated with topical oxygen include osteomylelitis, tendon and cartilage repair, sprains, fractures, burns and scalds, necrotizing fasciitis, pyoderma gangrenosum, refractory ulcers, diabetic foot ulcers and decubitus ulcers (bed sores) as well as cuts, abrasions, and surgically induced wounds or incisions.

10004] Wound dressings that provide topical oxygen therapy have been described. One such dressing is Oxyband™, which releases oxygen from an oxygen reservoir within the dressing. However, gaseous oxygen is trapped within the multilayers of the Oxyband^IM dressing and can escape the dressing prior to application. Another is Oxyzyme^I , a hydrogel dressing that produces oxygen within the dressing upon initiation of a complex series of (bio)chemical reactions, and then permits the transport of dissolved oxygen through the dressing to the wound surface. However, this dressing also potentially delivers hydrogen peroxide to the wound, which can be cytotoxic. Another oxygenated dressing is Oxygenesys^{1 M}, which is a closed-foam hydrogel made from polyacrylamide polymer that incorporates gaseous oxygen into the closed cells during the manufacturing process, where the embedded oxygen is delivered as dissolved oxygen to the wound. However, Oxygenesys^1M dressings rely on dissolution of the oxygen from gaseous bubbles, which is generally inefficient, and the fabrication route is complicated and expensive.

[0005] There is a need for improved wound dressings that provide topical oxygen to improve wound healing and the like. Alternatively, or in addition, there is a need for an alternative to known wound dressings that provide topical oxygen.

SUMMARY

[0006] The present disclosure arises from the present inventors finding that oxygen-loaded zeolites that release adsorbed oxygen when in contact with a wound provide an alternate means for accelerating wound healing.

[0007] According to a first aspect of the present disclosure, there is provided a wound dressing comprising an oxygen-loaded zeolite, wherein the oxygen-loaded zeolite releases oxygen when in use.

[0008] Zeolites are a family of microporous, aluminosilicate minerals, typically aluminosilicates of sodium, potassium, magnesium, calcium, and barium. Zeolites are able to bind cations and other molecules, including oxygen, within their porous structure. Zeolites have been used as cation exchangers, adsorbents, catalysts, molecular sieves and antibacterials. Zeolites have been used in medical technologies, and they have good biocompatibility. They have been used for drug delivery, tissue regeneration, detoxification, magnetic resonance imaging, skin whitening, and as hemostatic agents.

[0009] In certain embodiments, the oxygen-loaded zeolite of the first aspect is incorporated within a biopolymer matrix. In certain embodiments, the oxygen-loaded zeolite is incorporated within a biopolymer matrix comprising alginate and agarose. In certain embodiments, the oxygen-loaded zeolite releases dissolved oxygen when the wound dressing is applied to a wound. In certain embodiments, the released oxygen improves wound healing.

[0010] In certain embodiments, the wound dressing comprising an oxygen-loaded zeolite comprises a layer of oxygen-loaded zeolites incorporated within a biopolymer scaffold, wherein the layer has been lyophilised and then loaded with oxygen.

[001 1 ] In certain embodiments, the oxygen-loaded zeolite is selected from the group consisting of Zeolite 13X, fluorinated Zeolite Y, NaX zeolite, NaY zeolite, Faujasites, Zeolite Socony Mobil-5 (ZSM- 5), MFI type zeolites, and mordenites. In certain embodiments, the oxygen-loaded zeolite is Zeolite 13X. [0012 ] In certain embodiments, the wound dressing is in a form selected from a composite dressing, a hydrogel dressing, a foam dressing, hydrocolloid dressing, absorbent dressing, gelling fibre dressing, hydroselective dressing or an alginate dressing.

[0013] In certain embodiments, the wound is selected from a bum, a chronic wound, a hypoxic wound, a venous leg ulcer, a diabetic foot ulcer, a laceration, and an incision. In certain embodiments, the wound is hypoxic.

[0014] In certain embodiments, the zeolite is provided within the dressing at a concentration between 0.001 mg/cm³ and 1000 mg/cm³. In certain embodiments, altering the concentration of the oxygen- binding zeolite within the wound dressing correlates with the amount of oxygen released.

[0015] According to a second aspect of the present disclosure, there is provided a method of producing an oxygen-loaded zeolite layer for the wound dressing of the first aspect, the method comprising:

(a) providing an oxygen-binding zeolite suspension comprising an oxygen-binding zeolite;

(b) providing a biopolymer mixture;

(c) mixing the oxygen-binding zeolite suspension with the biopolymer mixture to produce an oxygen-binding zeolite biopolymer mixture;

(d) applying the oxygen-binding zeolite biopolymer mixture to a mould to form an oxygen- binding zeolite biopolymer layer;

(e) lyophilising the oxygen-binding zeolite biopolymer layer to form a lyophilised oxygen- binding zeolite biopolymer layer; and

(f) loading the lyophilised oxygen-binding zeolite biopolymer layer with oxygen.

[0016] In certain embodiments, step (e) comprises freezing the oxygen-binding zeolite biopolymer layer and then lyophilising the oxygen-binding zeolite biopolymer layer.

[0017] In certain embodiments, step (d) comprises applying the oxygen-binding zeolite biopolymer mixture to a structural layer within a mould to form an oxygen-binding zeolite biopolymer layer.

[0018] According to a third aspect of the present disclosure, there is provided a use of the wound dressing comprising an oxygen-loaded zeolite of the first aspect, or the use of the oxygen-loaded zeolite layer for the wound dressing produced by the method of the second aspect. In certain embodiments, the use is to improve wound healing.

[0019] According to a fourth aspect of the present disclosure, there is provided a use of an oxygen- loaded zeolite, wherein when in use, the oxygen-loaded zeolite releases oxygen to improve wound healing.

[0020] In certain embodiments, the oxygen-loaded zeolites are prepared by

(a) dispersing a predetermined amount of an oxygen-binding zeolite in a solution to produce an oxygen-binding zeolite suspension;

(b) lyophilising the oxygen-binding zeolite suspension; and

(c) exposing the oxygen-binding zeolite suspension to oxygen to form oxygen-loaded zeolites.

[0021 1 In certain embodiments, step (a) further comprises adding a biopolymer that provides a biological scaffold to the oxygen-binding zeolite suspension. In certain embodiments, the wound is hypoxic.

BRIEF DESCRIPTION OF FIGURES

[0022] Embodiments of the present disclosure will be discussed with reference to the accompanying drawings wherein:

[0023] Figure 1 provides scanning electron microscopy (SEM) images of zeolite dressings containing (a) 0.5g/g (AAZ1 ), (b) 5g/g (AAZ2), and (c) l Og/g (AAZ3) Zeolite 13X/biopolymer (Scale bar: 200 μηι, magnification: 100 x) and pie graphs showing dehydrated pore size distribution of dressings containing (d) 0.5g/g (AAZl), (e) 5g/g (AAZ2), and (f) l Og/g (AAZ3) Zeolite 13X/biopolymer;

[0024] Figure 2 provides an SEM image of 5g/g (zeolite/biopolymer) Zeolite 13X loaded composite dressings (AAZ2), with arrows indicating that zeolites are distributed throughout the dressing (scale bar: 200 μιη: magnification: 100 x);

[0025] Figure 3 provides SEM images of (a) zeolites alone at high magnification (Scale bar: 20 μιη, magnification: 1000 x); and zeolites impregnated on the surface of AAZ3 at (b) low magnification (Scale bar: 100 μιη, magnification: 200 x) and (c) high magnification (Scale bar: 20 μηι, magnification: 1000 x); [0026] Figure 4 provides graphical representations showing the compression modulus of the dressings containing 0, 0.5, 2.5, 5, 10 or 20g/g zeolite/biopolymer in the (a) dry state, and (b) swollen state following 24 hr in cell culture medium;

[0027] Figure 5 provides graphical representation of the swelling index of the dressings in water at 1 , 10 and 15 days, containing 0, 0.5, 2.5, 5, 10 or 2()g/g zeolite/biopolymer;

[0028] Figure 6 provides graphical representation of oxygen release from materials in deoxygenated MilliQ water, wherein the materials were (a) mock control (no addition), (b) dressing with 0 g/g zeolite/biopolymer (AA), (c) dressing with 0.5 g/g zeolite/biopolymer (AAZI ), or (d) an equivalent amount of zeolites alone as present in the dressing with 0.5 g/g zeolite/biopolymer;

[0029] Figure 7 provides graphical representation of oxygen release from dressings in deoxygenated MilliQ water with 0 g/g (AA), 0.5 g/g (AAZI ), 5 g/g (AAZ2) and lOg/g (AAZ3) zeolite/biopolymer;

[0030] Figure 8 provides Hoechst stained images of (a) 3T3 cells exposed to cell medium alone, (b) cells exposed to extracts from dressings with 0 g/g zeolite/biopolymer (AA), and (c) cells exposed to extracts from dressings with 0.5 g/g zeolite/biopolymer (AAZI );

[003 1 ] Figure 9 provides fluorescent images of FDA/PI stained cells of positive control cells (dead cells; top), negative control (live cells, middle) and cells incubated in the presence of extracts of dressing with 0.5 g/g zeolite/biopolymer;

[0032] Figure 10 provides a graphical representation of cell viability of 3T3 fibroblasts in an MTT assay with Zeolite- 13X at various concentrations, with cell viability relative to negative (no zeolite) control;

[0033] Figure 1 1 provides a graphical representation of cell viability of 3T3 fibroblasts in an MTT assay incubated for 24 h with extracts from dressings with 0, 0.5, 10 or 2()g/g zeolite/biopolymer;

[0034] Figure 12 provides fluorescent micrographs of images of FDA stained 3T3 mouse fibroblasts under (a) normoxic conditions; (b) hypoxic conditions; (c) hypoxic cells further treated with media alone and incubated for 24 h; and (d) hypoxic cells further treated with media containing the extracts from dressings containing 0.5 g/g zeolite/biopolymer and incubated for 24 h;

[0035] Figure 13 provides phase contrast (upper) and fluorescent (lower) micrograph images of human dermal fibroblasts under normoxia, hypoxia and hypoxic cells then exposed to media containing extracts from dressings containing 0.5 g/g zeolite/biopolymer; [0036] Figure 14 provides micrographs showing Hacat cells under normoxia in a wound scratch assay in either control medium or dressing containing 0.5 g/g zeolite/biopolymer conditioned media for 0, 24 and 48 h;

[0037] Figure 15 provides micrographs showing human dermal fibroblast cells under hypoxia in a wound scratch assay in either control medium or dressing containing 0.5 g/g zeolite/biopolymer conditioned media for 0, 24 and 48 h;

[0038] Figure 16 provides a graphical representation of dissolved oxygen release from wound dressing samples after: (a) 6 months storage (Batch 3); (b) 3 months storage (Batch 2); and (c) 0 months storage (Batch 1 );

[0039] Figure 17 provides macroscopic pictures (left) of partial thickness wounds treated with T, CI and C2 wound dressings after 4, 8, 10 and 12 days, and a graphical representation (right) of the reduction in wound area for wounds treated with T, C 1 and C2 wound dressings after 12 days (all data reported as mean ± SD, * p < 0.05 (n=4));

[0040] Figure 18 provides micrographs showing the immunohistochemistry of wounds after 4 days. The panel displays staining against CD68 (macrophage marker) for wounds treated with (a) T, (b) C I and (c) C2 and (d-f) represent their higher magnifications. Macrophages are stained brown; and

[0041 1 Figure 19 provides micrographs showing histology of the wounds treated with T, C I and C2 after 12 days stained with Masson's trichrome. Arrows highlight wound area remaining.

DESCRIPTION OF EMBODIMENTS

[0042] A suitable level of oxygenation of wounds has been associated with improved wound healing. In many chronic wounds, there is a prolonged undersupply of oxygen to the tissue (hypoxia). This may lead to a slowing or stagnation of wound healing processes, since numerous molecular processes are dependent on a sufficient supply of oxygen or are induced by reactive oxygen species (ROS).

[0043] As disclosed herein, oxygen-loaded zeolites that release adsorbed oxygen can increase the dissolved oxygen concentration within a solution. Additionally, the present inventors have shown that under hypoxic conditions, cell growth is improved when the cells are incubated in the presence of culture media that has previously been conditioned with dressings containing oxygen-loaded zeolites. Moreover, in an in vitro wound scratch test performed under hypoxic conditions, proliferation and migration of cells was significantly improved when incubated in media conditioned with dressings containing oxygen- loaded zeolites. This scratch test provides a suitable model for in vivo hypoxic wound healing. Moreover, as disclosed herein, the amount of oxygen that can be released from the oxygen-loaded zeolites can be altered by altering the concentration of the zeolites applied to the dressing. Accordingly, oxygen-loaded zeolites in the context of a biological scaffold or wound dressing may increase the oxygen content within the wound environment and assist wound healing. Further, dressings containing oxygen-loaded zeolites may also be used in other applications where increased oxygen levels would be beneficial for the growth or preservation of cell and/or tissues. For instance, the dressing could be used to provide additional oxygen to cells during cell culture or to cells and/or tissues exposed to hypoxic conditions during transport (eg, pancreatic islets). The technology disclosed herein involves a simple fabrication procedure. Further, the oxygen adsorbed within the pores of the scaffold-bound zeolites can be efficiently released into the surrounding solution.

[0044] In a first aspect, provided herein is a wound dressing comprising an oxygen-loaded zeolite, wherein the oxygen-loaded zeolite releases oxygen when in use. In certain embodiments, the increase in oxygen concentration improves wound healing or assists with cell growth, tissue preservation or other biochemical processes.

[0045 ] As would be understood by a person skilled in the art, the term "zeolite" refers to a microporous, crystalline form of aluminosilicate, commonly of sodium, potassium, calcium, magnesium and/or barium. The framework structure of a zeolite contains pores (also referred to as intracrystalline channels or interconnected voids) that can bind and trap other small molecules (eg by adsorption), commonly cations, water molecules and gases. The small molecules that can be trapped by a zeolite are generally smaller than the pore size of the zeolite. Accordingly, zeolite structure, notably the pore size, may dictate what molecules bind in the pores. The pore size of zeolites is typically less than 2 nm (20 A) in diameter.

[0046 ] Zeolites may have the ability to be dehydrated and rehydrated without experiencing significant changes in their crystalline structure. A zeolite can be artificially synthesised or found in nature (ie, are mined). Chemically, they are represented by the empirical formula:

M_{2 η}Ο· A1₂0₃ · vSi0₂ · wH₂0 (I) where y is 2 - 200,

M is the cation balancing the charge of the aluminosilicate ion n is the cation valence, and w represents the water contained in the voids of the zeolite. [0047] Structurally, zeolites are complex, crystalline inorganic polymers based on an infinitely extending three-dimensional, four-connected framework of A10₄ and Si0₂ tetrahedrally linked to each other by the sharing of oxygen ions. Each A10₄ tetrahedron in the framework bears a net negative charge which is balanced by an extra framework cation. At least 40 naturally occurring zeolites have been discovered, and at least 150 synthetically created zeolites are known. Synthetic zeolites can be manufactured in very precise and uniform sizes having a uniform pore size, and can be selected for a particular application on the basis of their structure and pore size.

[0048 ] The terms "oxygen-loaded zeolite" and "oxygen-binding zeolite" as used herein refer to a zeolite that is capable of binding or adsorbing oxygen. These terms can be interchangeably used to refer to the same zeolites; however, the term "oxygen-loaded zeolite" is intended to refer to a zeolite that has oxygen bound within its pores. In certain embodiments, the oxygen-loaded zeolite has undergone a specific step that removes trapped gases, water, etc, from a significant majority of its pores, and is then exposed to a high concentration of oxygen (ie oxygen loading) such that a significantly high percentage of the pores of the zeolite contain bound oxygen. The oxygen-binding zeolites of the present disclosure may be any zeolite known in the art, providing that the resulting zeolite is capable of binding oxygen and is not overly detrimental to wound healing or other biochemical processes. In certain embodiments, the oxygen- binding zeolite is not cytotoxic. An oxygen gas molecule, dioxygen (0₂) is approximately 2.8 A (0.28 nm) in size.

[0049 ] In certain embodiments, the oxygen-binding zeolite of the present disclosure may selectively bind oxygen over other molecules; however, in another embodiment, the oxygen-binding zeolites of the present disclosure may bind oxygen and other molecules (ie in the presence of oxygen and other molecules). Zeolites that may be oxygen-loaded include, but are not limited to, Y type Zeolites (NaY type zeolites, fluorinated Zeolite Y), X type zeolites (including Zeolite 13X, NaX zeolites, Na-CeX zeolites, CaX, CaLSX, LiLSX, alkaline-earth-metal-cation-exchanged Zeolite X), or A type zeolites (NaA or 4A, 5A and CaA zeolites). Other useful oxygen-binding zeolites are Faujasites (X and Y type zeolite), Zeolite Socony Mobil-5 (HZSM-5, Cu/HZSM5), MFI type zeolites, alkylperfluorinated zeolites and mordenites (Na, Ca and Ba mordenites). In certain embodiments, the zeolite is a modified zeolite, eg., a surfactant modified zeolite, surface modified zeolite, etc In certain embodiments, the oxygen-loaded zeolite is selected from the group consisting of Zeolite 13X, fluorinated Zeolite Y, NaX zeolite, NaY zeolite, faujasites, Zeolite Socony Mobil-5 (ZSM-5), MFI type zeolites, and mordenites.

[0050 ] In certain embodiments, the oxygen-binding zeolite has a pore size that is greater than 2.8 A (0.28 nm). In certain embodiments, the oxygen-loaded zeolite has a pore size of less than 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, or 3 A (ie, 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, or 0.3 nm). In certain embodiments, the pore size of the oxygen-binding zeolite is between 3 A and 15A (0.3 and 1.5 nm). In certain embodiments, the pore size of the zeolite is between 9 and 12 A (0.9 and 1.2 nm). In certain embodiments, the pore size of the zeolite is between 7 and 1 1 A (0.7 and 1.1 nm). In certain embodiments, the pore size of the zeolite is between 4 and 6 A (0.4 and 0.6 nm). In certain embodiments, the pore size of the zeolite is between 6 and 9 A (0.6 and 0.9 nm. As would be understood by a person skilled in the art, 1 A is equivalent to 0.1 nm.

[0051 ] In certain embodiments, the oxygen-binding zeolite may be Zeolite 13X. Zeolite 13X has an effective pore diameter of approximately 10 A in size, and is the sodium form of the type X zeolite. However, in the presence of some cations, the pore size of Zeolite 13X is approximately 8 A in size. Zeolite 13X has the formula:

Na₂0: 1 A1₂0₃ : 2.8 ± 0.2 Si0₂ : χΗ,Ο

[0052 ] In certain embodiments, the oxygen-binding zeolite is Zeolite 5A. Zeolite 5A has an effective pore opening of about 10 A in size. Zeolite 5 A has the formula:

0.80 CaO : 0.20 Na₂0 : 1 A1₂0₃: 2.0 ± 0.1 Si0₂: x H₂0

[0053] Faujasite is a mineral group in the zeolite family of silicate minerals. The group consists of faujasite-Na, faujasite-Mg and faujasite-Ca. They all share the same basic formula by varying the amounts of sodium, magnesium and calcium:

(Na₂,Ca,Mg)3 ₅[Al₇Si₁₇0₄₈] -32(H₂0)

[0054] The pore of faujasites is formed by a 12-membered ring, has a diameter of approximately 7.4 A. The inner cavity has a diameter of 12 A. Depending on the silica-to-alumina ratio of their framework, synthetic faujasite zeolites are divided into X and Y zeolites. In X zeolites that ratio is between 2 and 3, while in Y zeolites it is 3 or higher. The negative charges of the framework are balanced by the positive charges of cations in non-framework positions.

[ 00551 The typical oxide formula of a NaX zeolite is Na₂0: 1 A1₂0₃ : 2.5Si0₂ : 6H₂0

[0056] ZSM-5, Zeolite Socony Mobil-5 (framework type MFI from ZSM-5 (five)), is an aluminosilicate zeolite belonging to the pentasil family of zeolites. Its chemical formula is:

Na_nAl_nSi_{96 ll}0₁₉₂- 16H₂0 (0<n<27). [0057] An example of a ZSM-5 zeolite is also known as a MF1 zeolite, having a pore structure between 5 and 7 A.

[0058] Mordenite is a zeolite mineral with the chemical formula: (Ca, Na₂, K₂)Al₂Si₁₀O₂4-7H₂O.

[0059] In certain embodiments, an oxygen-binding zeolite (e.g., such as Zeolite 13X) is dispersed in a suitable solution to form an oxygen-binding zeolite suspension. In certain embodiments, the solution may be water. However, other suitable solutions known to persons skilled in the art may be used, such as phosphate buffered saline, or other biocompatible solutions. The zeolite may be added to the solution in any suitable concentration.

[ 0060] In certain embodiments, the oxygen-binding zeolite suspension is sonicated. The sonication step may break down agglomerates of the zeolites to provide a uniform suspension, and may "activate" the zeolites. The sonication conditions used may be any suitable conditions known to persons skilled in the art, providing that the technique used results in a generally uniform suspension.

[0061 ] In certain embodiments, the oxygen-binding zeolite suspension is mixed with a biopolymer in a suitable manner in order to incorporate the oxygen-binding zeolite within the biopolymer matrix, to produce an oxygen-binding zeolite biopolymer mixture. In certain embodiments, the biopolymer is a suitable biocompatible polymer(s) that can provide a biological scaffold. In certain embodiments, the biopolymer is selected from agarose, alginate, sodium carboxymethylcellulose, gelatin, pectin, elastomers, polyurethane, silicone, collagen, polylactic acid (PLA), chitin, chitosan, keratin, polycaprolactone, polyacrylonitrile, polyethylene, etc. In certain embodiments, the biopolymer is a combination of more than one biopolymer. In certain embodiments, the biopolymer is a mixture of agarose and alginate. In certain embodiments, the biopolymer is dissolved in an aqueous solution, and the oxygen-binding zeolite can be incorporated into the matrix of the biopolymer by combining the oxygen- binding zeolite suspension with the biopolymer solution and mixing the resulting zeolite biopolymer mixture, for example, with a magnetic stirrer. The biopolymer may be used at any suitable concentration known to persons skilled in the art. In certain embodiments, the biopolymer is used at a concentration between 0.01 % (w/v) and 10% (w/v); or between 0.1% (w/v) and 2% (w/v). In certain embodiments, the biopolymer is used at a concentration approximately 0.5% (w/v). In certain embodiments, the biopolymer is used at a concentration approximately 1.0% (w/v). In certain embodiments, the biopolymer comprises agarose used at a concentration between 0.01 % (w/v) and 10% (w/v); or between 0. 1% (w/v) and 2% (w/v). In certain embodiments, the agarose is used at a concentration approximately 0.5% (w/v). In certain embodiments, the agarose is used at a concentration approximately 1% (w/v). In certain embodiments, the biopolymer comprises alginate is used at a concentration between 0.01% (w/v) and 10% (w/v); or between 0.1 % (w/v) and 5% (w/v). In certain embodiments, the alginate is used at a concentration approximately 0.5% (w/v). In certain embodiments, the alginate is used at a concentration approximately 1 % (w/v).

[0062 ] In certain embodiments, the concentration of the zeolite within the oxygen-binding zeolite biopolymer mixture is between 0.01% w/v and 70% w/v. In certain embodiments, the concentration of the zeolite within the oxygen-binding zeolite biopolymer mixture is between 0.1% w/v and 50% w/v. In certain embodiments, the concentration of the zeolite within the oxygen-binding zeolite biopolymer mixture is between 1% w/v and 40% w/v. In certain embodiments, the concentration of the zeolite within the oxygen-binding zeolite biopolymer mixture is selected from 1% w/v, 2% w/v, 3% w/v, 4% w/v, 5% w/v, 6% w/v, 7% w/v, 8% w/v, 9% w/v, 10% w/v, 15% w/v, 20% w/v, 25% w/v, 30% w/v, 35% w/v or 40% w/v. In certain embodiments, the concentration of the zeolite within the oxygen-binding zeolite biopolymer mixture is between 0.01 g/ml and 0.4 g/ml, for example, 0.01 , 0.05. 0.1 , 0.2, 0.3, or 0.4 g/ml.

[0063] The oxygen-binding zeolite biopolymer mixture may be applied to a mould to form an oxygen- binding zeolite biopolymer layer. In certain embodiments, the mould may be any suitable item known to a person skilled in the art. The mould may shape the zeolite biopolymer into a desired shape, for example for a wound dressing, or for other uses. The mould may comprise tissue culture plastics (eg wells of 6, 12, 24, or 96 well plates), other cell culture plates, petri dishes, etc or other suitable items that are capable of moulding the zeolite biopolymer into a desired shape.

[0064 ] In certain embodiments, the oxygen-binding zeolite biopolymer mixture may be applied to a suitable structural layer to form at least part of the wound dressing comprising oxygen-binding zeolites. The structural layer could be a dressing layer, such as a gauze, woven and non-woven fibres, porous foams, or a film, etc, that is suitable for forming a layer of a wound dressing as described herein. In certain embodiments, the structural layer may be applied to the mould, and then the oxygen-binding zeolite biopolymer mixture may be applied to the structural layer within the mould to form a dressing layer comprising oxygen-binding zeolites.

[0065] As described herein, the oxygen-binding zeolite biopolymer layer may be lyophilised or dried. In certain embodiments, the wound dressing comprising oxygen-binding zeolites are dried or lyophilised prior to application to a wound. As described herein, zeolites can be dehydrated and re-hydrated without experiencing significant changes in their crystalline structure. In certain embodiments, the oxygen- binding zeolite biopolymer layer is dried, for example, air dried, but more preferably, in a suitable oven. In certain embodiments, the oxygen-binding zeolite biopolymer layer is lyophilised. In certain embodiments, the oxygen-binding zeolite biopolymer layer is freeze-dried. In certain embodiments, the wound dressing is lyophilised, or freeze-dried (noting that in some circumstances, the terms "lyophilisation" and "freeze-drying" can be used interchangeably, as will be understood by a person skilled in the art). In certain embodiments, the drying step is conducted under vacuum to remove entrapped gas, water, etc molecules from the pores of the zeolite. In certain embodiments, the drying step is followed by a vacuum step to remove entrapped gas, water, etc molecules from the pores of the zeolite. A lyophilisation or drying step may remove water, and other solution components, and/or trapped gases from the pores of the zeolite. This lyophilisation or drying step accordingly permits increased binding of oxygen to the zeolite pores. In certain embodiments, the lyophilisation or diying step removes all, or at least nearly all (ie a very high percentage), of the gases and or water or other molecules from the zeolite pore.

[0066] In certain embodiments, the lyophilised or dried oxygen-binding zeolite biopolymer layer (or wound dressing) is exposed to oxygen to form oxygen-loaded zeolites. The oxygen-binding zeolites may be loaded with oxygen using any suitable method known to persons skilled in the art. In certain embodiments, the lyophilised or dried wound dressing comprising oxygen-binding zeolites of the present invention is exposed to air in the atmosphere to produce an oxygen-loaded zeolite wound dressing. In certain embodiments, the lyophilised oxygen-binding zeolite wound dressing is exposed to a high concentration of oxygen to produce an oxygen-loaded zeolite wound dressing. In certain embodiments, the lyophilised oxygen-binding zeolite wound dressing is exposed to pure, or essentially pure oxygen (as would be understood by a person skilled in the art) to produce an oxygen-loaded zeolite wound dressing. In certain embodiments, the lyophilised oxygen-binding zeolite wound dressing is back filled with pure or essentially pure oxygen (as would be understood by a person skilled in the art). In certain embodiments, there is more than 70%, 80%, 90%, 95%, 97%, or 99% adsorption of oxygen in the zeolite pores (ie compared to other molecules in the pores). In certain embodiments, more than 70%, 80%, 90%, 95%, 97%, or 99% of the pores of the oxygen-loaded zeolite are adsorbed with oxygen.

[0067] In certain embodiments, the oxygen loading step occurs immediately after the lyophilisation or drying step, for example, by connecting an oxygen line to the freeze-drying apparatus, such that the vacuum created by the freeze-drier is back filled with oxygen. In an alternative embodiment, the oxygen loading step occurs subsequent to the lyophilisation or drying step, for example, by moving the oxygen- binding zeolite biopolymer layer from the freeze dryer to a vacuum drying tube and placing the layers or dressings under vacuum at a suitable temperature (ie below the degradation temperature of the biopolymer as would be understood by a person skilled in the art). After a suitable period under vacuum, the tube and dressings may be backfilled with oxygen. In certain embodiments, this process can be repeated multiple times to maximise or optimise oxygen loading of the zeolites. In certain embodiments, the wound dressing comprising oxygen-loaded zeolite comprises oxygen-loaded zeolites incorporated within a biopolymer scaffold, which is then lyophilised and loaded with oxygen. In certain embodiments, following freeze drying, the vacuum in the freeze drying systems is back filled with atmospheric gases. In certain embodiments, following freeze drying, the vacuum in the freeze drying systems is back filled with atmospheric gases, and the samples are transferred to a heated vacuum tube, evacuated for a time and then backfilled with oxygen. The dressings are then sealed in an air tight manner (eg in bags) under oxygen. In certain embodiments, following freeze drying, the vacuum in the freeze dryer is directly backfilled with oxygen. The dressings are then sealed in an air tight manner (eg in bags) under oxygen.

[0068 ] In certain embodiments, the oxygen-loaded zeolite wound dressing is sealed in an air tight manner and stored until required, to prevent loss of oxygen to the atmosphere or exchange with other gases, such as nitrogen. In certain embodiments, the oxygen-loaded zeolite wound dressing is sealed in an air tight manner under an oxygen blanket and stored until required. Storage of the wound dressing in an air tight manner may essentially prevent loss of oxygen from the zeolites into the atmosphere during storage.

[0069 ] In certain embodiments, the lyophilised oxygen-binding zeolite wound dressing is stored until required, and then exposed to a high concentration of oxygen to produce an oxygen-loaded zeolite wound dressing immediately prior to use. In certain embodiments, the lyophilised oxygen-binding zeolite wound dressing is stored (for example, in air tight packaging) until required, and then exposed to essentially pure oxygen to produce an oxygen-loaded zeolite wound dressing immediately prior to use.

[0070J In certain embodiments, the oxygen-binding zeolite wound dressing is dried prior to storage, and when required, is placed under vacuum at a suitable temperature to remove adsorbed gases etc, and is then exposed to oxygen as described herein, for example, immediately prior to use, or alternatively, is stored for a further time in air tight packaging.

[ 0071 ] In certain embodiments, the dressings may be irradiated or sterilised by appropriate means.

[0072] In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen- binding zeolite biopolymer mixture is between 0.05 g/g polymer and 200 g/g polymer. In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer mixture is between 0. 1 g/g polymer and 40 g/g polymer. In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer mixture is between 0.5 g/g polymer and 20 g/g polymer.

[0073 ] As described herein, the oxygen-binding zeolite biopolymer mixture may be added to a mould, prior to the lyophilisation step. As would be understood by a person skilled in the art, the concentration of the zeolite per cm² of the resulting dressing or layer will vary with the volume of the oxygen-binding zeolite biopolymer mixture added to the mould per cm² of the mould. Accordingly, in certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.001 mg/cm² and 1000 mg/cm². In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.01 mg/cm² and 100 mg/cm². In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.1 mg/cm² and 50 mg/cm². In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.55 mg/cm² and 22 mg/cm². As would be understood by a person skilled in the art, the concentration of the zeolite (eg in mg/cm²) within the lyophilised or dried oxygen-binding zeolite biopolymer layer may be the same concentration as provided within the dressing.

[0074 ] In certain embodiments, when oxygen-binding zeolite biopolymer layer is freeze dried to afford the dressing, the zeolite mass per gram of biopolymer is in the range of 0.5 to 20 g/g. In certain embodiments, the wound dressings comprising the oxygen-loaded zeolites, the mass of the zeolite per surface area is between 0.00055 and 0.022 g/cm². However, this mass per area can change depending on the volume and area of the dressing as described herein.

[ 0075] In certain embodiments, the concentration of the zeolite is measured as mass per volume of the dried dressing. As would be understood by a person skilled in the art, the concentration of the zeolite per cm³ of the resulting dressing or layer may vary dependent upon the mass of the zeolite used to prepare the dressing. Accordingly, in certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.001 mg/cm³ and 1000 mg/cm³. In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.01 mg/cm³ and 100 mg/cm³. In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.1 mg/cm³ and 50 mg/cm³. In certain embodiments, the concentration of the zeolite within the lyophilised or dried oxygen-binding zeolite biopolymer layer is between 0.42 mg/cm³ and 22 mg/cm³. As would be understood by a person skilled in the art, the concentration of the zeolite (eg in mg/cm³) within the lyophilised or dried oxygen-binding zeolite biopolymer layer may be the same concentration as provided within the dressing.

[0076] In certain embodiments, when the lyophilised oxygen-loaded zeolite dressing disclosed herein is exposed to a solution (eg in a wound environment or in other biological conditions, including tissue culture), the zeolites release the oxygen as dissolved oxygen into the solution.

[0077] As used herein, the term "wound" is intended to refer to an interruption in the integrity of the skin. The wound may range from minor to life threatening, and may be classified as open or closed; acute or chronic; clean, contaminated or infected; internal or external.

[0078] Acute wounds typically heal uneventfully (without serious complications), generally within a predictable amount of time. However, chronic wounds may take a longer time to heal and might have significant complications. Clean wounds have no foreign materials or debris inside, whereas contaminated wounds or infected wounds might have dirt, fragments of the causative agent, bacteria or other foreign materials. Internal wounds may result from impaired immune and nervous system functions and/or decreased supply of blood, oxygen or nutrients to that area; such as in cases of chronic medical illness (diabetes, atherosclerosis, deep vein thrombosis, etc). External wounds are usually caused by penetrating objects (deep abrasions, incisions, and lacerations) or non-penetrating trauma (e.g., blunt trauma, friction, abrasions, lacerations, contusions, concussions etc) and other miscellaneous causes (including thennal burns, chemical wounds; bites and/or stings; electrical wounds; etc). In certain embodiments, the wound is an open wound. In certain embodiments, the wound is an external wound. In certain embodiments, the wound is a chronic wound. In certain embodiments, the wound is hypoxic. The wound may be selected from the group consisting of incisions, lacerations, abrasions, contusions, punctures, trauma, burns, chemical wounds, and electrical wounds. In certain embodiments, the wound may be selected from wounds associated with osteomyelitis, tendon and cartilage repair, sprains, fractures, burns and scalds (including first degree burns, second degree burns, third degree burns), necrotizing fasciitis, pyoderma gangrenosum, refractory ulcers, diabetic foot ulcers, venous leg ulcers, and decubitus ulcers (bed sores) as well as cuts, abrasions, and surgically induced wounds or incisions.

[0079] Hypoxia is a reduction in oxygen delivery below tissue demand. A hypoxic wound, as used herein is intended to refer to a wound that has a lower tissue partial pressure of oxygen (p02) compared to the p02 of the same tissue (or an appropriately adjusted value thereof) under healthy conditions in vivo. Hypoxic wounds tend to heal poorly, and can benefit from topical oxygen therapy. Wound hypoxia can be tested using any test known to those skilled in the art, including by measuring transcutaneous oxygen tension (Ptc02), ankle brachial index, skin perfusion pressure, laser Doppler flow, transcutaneous oximetry (Ptc02 or TCOM). One study found that a chronic wound tissue may have a p0₂ of 5-20 mmHg as compared with 30-50 mmHg in control tissue (Sheffield, 1988). In certain embodiments, a hypoxic wound may have a wound p02 of less than 40 mmHg. In certain embodiments, a hypoxic wound may have a wound p02 of less than 30 mmHg. In certain embodiments, wounds that are not hypoxic may have wound p02 of between 40-50 mmHg.

[0080 ] As used herein, the term "wound dressing" is intended to refer to one or more layers that may be applied to a wound. The wound dressing may have a protective barrier function, for example, that acts as a skin substitute, and it may absorb wound exudate, and is typically sterile. Ideally, a wound dressing creates an environment that facilitates wound healing. Wound dressings that create and maintain a moist environment may provide enhanced conditions for wound healing. However, the presence of excess exudate at the site of a wound may be detrimental, for example, by decreasing oxygen at the site. The wound dressing may be absorbent, and absorb the wound exudate. In certain embodiments, it may maintain the wound in a moist environment. A wound dressing may be applied directly in contact with a wound. In certain embodiments, the wound dressing may be indirectly in contact with the wound dressing.

[0081 ] There are various types of wound dressings. The wound dressing disclosed herein may be any suitable type of wound dressing, providing it is possible to suitably apply or impregnate the dressing with oxygen-binding zeolites. In certain embodiments, the wound dressing may be in the form of a composite dressing, foam dressing, a gauze dressing, an alginate dressing, a hydrocolloid dressing, an antimicrobial dressing, a semipermeable film dressing, hydrogel dressing, a film dressing, absorbent dressing, gelling fibre dressing, hydroselective dressing, etc. In certain embodiments, the wound dressing is a composite dressing.

[0082 ] A composite dressing is a multi-layer dressing that may combine different types of dressing features into a single dressing, eg, a waterproof layer, an absorptive layer, a strike-through barrier. In certain embodiments, a composite dressing may comprise an inner layer comprising a non-adherent layer, an absorptive middle layer, and a water-proof and/or air tight outer layer. In certain embodiments, the wound dressing disclosed herein is a composite dressing comprising oxygen-loaded zeolite incorporated within a biopolymer matrix that has been applied to a suitable structure (eg gauze bandage, a film, a transparent adhesive film, an acrylic adhesive, a moisture vapour permeable layer, waterproof layer etc) that is suitable for forming a layer of a wound dressing as described herein. In certain embodiments, the composite dressing prevents gas exchange and may optionally provide a means of adhering the wound dressing to the patient as would be well understood by a person skilled in the art.

[0083 ] A semipermeable film may comprise sterile plastic sheets of polyurethane coated with hypoallergenic acrylic adhesive and are used mainly as a transparent primary wound cover. They may be flexible, and may be impermeable to fluids and bacteria, but permeable to air and water vapour, and may maintain a moist wound environment.

[0084 ] In hydrocolloid films, sodium carboxymethylcellulose, gelatin, pectin, elastomers, adhesives, etc are bonded to a carrier of semipermeable film or a foam sheet to produce a flat, occlusive, adhesive dressing that forms a gel on the wound surface, promoting moist wound healing. Cross linkage of the materials used may influence the viscosity of the gel under the dressing. Hydrocolloids may be impermeable to water vapour and air and may be used to rehydrate a wound. Hydrocolloid fibres are available in the form of a hydrophilic, non-woven flat sheet, referred to as hydrofibre dressings. On contact with exudates, fibres are converted from a dry dressing to a soft coherent gel sheet, making them suitable for wounds with a large amount of exudate.

[0085 ] Hydrogel dressings may comprise a matrix of cross-linked polymers with up to 99 % water content enabling them to donate water molecules to the wound surface and to maintain a moist environment at the wound bed. The polymers may be only partially hydrated, and accordingly, the hydrogels may have the ability to absorb a degree of wound exudate. They may transmit moisture vapour and/or oxygen, but their bacterial and fluid permeability may be dependent upon the type of secondary dressing used. In certain embodiments, hydrogel dressings may be amorphous (gels), impregnated (where the hydrogel is saturated onto a gauze pad, nonwoven sponge ropes and/or strips, etc), or as a sheet (where the hydrogel supported by a thin fibre mesh).

[0086] Alginate dressings comprise alginates that may be produced from the naturally occurring calcium and sodium salts of alginic acid found in a family of brown seaweed (Phaeophyceae). They generally fall into one of two kinds: those containing 100% calcium alginate or those that contain a combination of calcium with sodium alginate, usually in a ratio of 80:20. Alginates partly dissolve on contact with wound fluid to form a hydrophilic gel as a result of the exchange of sodium ions in wound fluid for calcium ions in the dressing. Alginates can absorb 15 to 20 times their weight of fluid, making them suitable for highly exuding wounds. They may be less suitable for use on wounds with little or no exudate as they may adhere to the healing wound surface.

[0087] Foam dressings may comprise a foam fonned from a polyurethane or silicone, etc. They transmit moisture vapour and oxygen and provide thermal insulation to the wound bed. They may facilitate uniform dispersion of exudate throughout the absorbent layer and prevent exterior leakage (strike- through) due to the presence of a semipermeable backing.

[0088] In certain embodiments, the wound dressing is at least partially impermeable to gas exchange, to retain the released oxygen in the wound environment. In certain embodiments, the wound dressing comprising the oxygen-loaded zeolites provides a suitable barrier to protect the wound and/or retain the released oxygen within the wound environment, in the absence of a secondary wound dressing. However, in an alternative embodiment, the wound dressing disclosed herein may be used in conjunction with at least one secondary wound dressing. In certain embodiments, an at least one secondary dressing may provide an outer layer, that is, lie over the wound dressing of this disclosure, for example to provide a barrier function, or provide a means to adhere the wound dressing comprising the oxygen-loaded zeolites to the patient. In another embodiment, an at least one secondary dressing may provide an inner layer to the wound dressing of the present disclosure, that is, lie under the wound dressing of the present disclosure, for example so that the secondary dressing is in direct contact with the wound. This may allow for a secondary wound dressing that has suitable properties be in direct contact with the wound. In this arrangement, it would be necessary for the secondary bandage to be at least partially permeable to the oxygen released by the oxygen-loaded zeolites.

[0089] A moist wound environment may assist healing. However, excess wound exudate is generally detrimental to wound healing. Further, these criteria may be affected by the characteristics of a particular wound. In certain embodiments, the lyophilised or dried wound dressings may be applied directly to the wound. In certain embodiments, at least some of the exudate of the wound is absorbed by the zeolites and/or by the biocompatible polymer. In certain embodiments, the wound dressings may be at least partially rehydrated prior to being applied to a wound.

[0090 ] As disclosed herein, the oxygen-loaded zeolites of the disclosure can increase the dissolved oxygen in a solution. Accordingly, in certain embodiments, when applied to a wound, the oxygen-loaded zeolites release oxygen. In certain embodiments, the released oxygen improves wound healing. Advantageously, the oxygen-loaded zeolites release oxygen over a sustained period. The sustained period may be from about 3 hours to about 20 hours. Where there is sufficient moisture within the wound dressing environment (i.e., when applied to the wound), the released oxygen may be dissolved oxygen. Accordingly, in certain embodiments, the released oxygen increases the dissolved oxygen concentration within the wound dressing. Further, in certain embodiments, the oxygen-loaded zeolites may increase the dissolved oxygen concentration at the surface of the wound. The presence of the oxygen-loaded zeolites within the wound dressing may therefore enhance wound healing.

[0091 ] However, the oxygen-loaded zeolites disclosed herein may also assist in other biochemical processes, such as improved cell growth, tissue preservation (eg during cells or tissue transport), etc to increase the oxygen available to cells, for example under low-oxygen or hypoxic conditions. In certain embodiments, the oxygen-loaded zeolites, or an extract derived from the oxygen-loaded zeolites, can be added to cells or tissues within a solution containing the cells or tissues, or within a solution applied to the tissues or cells, to improve the dissolved oxygen content within the solution. In certain embodiments, the oxygen-loaded zeolites, or an extract derived from the oxygen-loaded zeolites, can be added to cells or tissues within a container (ie an air tight container) in which the cells or tissues are stored, as would be understood by a person skilled in the art. One embodiment is the use of oxygen loaded zeolites of the present disclosure during the transport of cells or tissues intended for transplantation.

[ 0092] As described herein, the present inventors have realised that the amount of oxygen released by a dressing (eg of a constant size) can be altered by altering the concentration of the oxygen-loaded zeolite in the dressing. In particular, increasing the concentration of the oxygen-binding zeolite in the zeolite suspension increases the amount of oxygen that the oxygen-loaded zeolite dressing is able to release, for example, when applied to a wound.

[0093] The wound dressing may be applied to a suitable wound using methods known to those skilled in the art. [0094 ] The present disclosure also provides a method of producing the wound dressing described herein. Accordingly, in an aspect, the present disclosure provides a method of producing a wound dressing comprising oxygen-loaded zeolites for improving wound healing, the method comprising:

(a) providing a suitable oxygen-binding zeolite suspension comprising an oxygen-binding zeolite;

(b) providing a biopolymer mixture;

(c) mixing the oxygen-binding zeolite suspension with a biopolymer mixture to produce an oxygen-binding zeolite biopolymer mixture;

(d) applying the oxygen-binding zeolite biopolymer mixture to a mould to form an oxygen- binding zeolite biopolymer layer; and

(e) lyophilising the oxygen-binding zeolite biopolymer layer; and

[0095 ] In certain embodiments, the oxygen-binding zeolite suspension of step (a) comprises a predetermined amount or concentration of the oxygen-binding zeolite. In certain embodiments, the oxygen-binding zeolite suspension of step (a) is sonicated. In certain embodiments, step (e) comprises freezing the oxygen-binding zeolite biopolymer layer of step (d) and then lyophilising the oxygen-binding zeolite biopolymer dressing.

[0096] In certain embodiments, step (d) comprises applying the oxygen-binding zeolite biopolymer mixture to a structural layer within a mould to form an oxygen-binding zeolite biopolymer layer, for example, a gauze, woven and non-woven fibres, porous foams, or a film, etc that is suitable for forming a layer of a wound dressing as described herein, etc, that is suitable for forming a layer of a wound dressing as described herein.

[0097] In an aspect, the oxygen-binding zeolite suspension can be incorporated within a preformed fibrous scaffold, foam or porous matrix known to those skilled in the art to fonn an oxygen-binding zeolite biopolymer dressing.

[0098] In an aspect, the oxygen-binding zeolite suspension with a biopolymer mixture can be mixed and applied directly to a mould to form an oxygen-binding zeolite biopolymer dressing layer for incorporation within a wound dressing, for example, a layer within a composite dressing. [0099 ] As detailed herein, the present inventors have shown that preparing a wound dressing using the method disclosed herein may result in the oxygen-loaded zeolites being distributed throughout the wound dressing. In certain embodiments, the wound dressing is impregnated with the oxygen-loaded zeolites.

[00100] In a further aspect, the present disclosure provides a method of producing an oxygen- loaded zeolite layer for the wound dressing of any one of the preceding claims, the method comprising the following steps:

(b) providing a biopolymer mixture;

(e) lyophilising the oxygen-binding zeolite biopolymer layer; and

(f) exposing the oxygen-binding zeolite biopolymer layer to oxygen to form the oxygen- loaded zeolite layer for the wound dressing.

[00101 ] In certain embodiments, step (d) comprises applying the oxygen-binding zeolite biopolymer mixture to a structural layer within a mould to form an oxygen-binding zeolite biopolymer layer, for example, for example, a gauze bandage, or a film, etc, that is suitable for forming a layer of a wound dressing as described herein.

[00102] In certain embodiments, step (e) comprises freezing the oxygen-binding zeolite biopolymer layer and then lyophilising the oxygen-binding zeolite biopolymer layer.

[00103 ] In certain embodiments, the features of the wound dressing described herein may also be features of the oxygen-loaded zeolite layer for the wound dressing, as appropriate.

[00104] In a further aspect, the present disclosure provides a use of the wound dressing comprising an oxygen-loaded zeolite described herein. In an aspect, the present disclosure provides a use of the wound dressing produced by the method of producing an oxygen-loaded zeolite layer for the wound dressing as described herein. In certain embodiments, the use is to improve wound healing. [00105] As will be appreciated by person skilled in the art, the oxygen-loaded zeolite may potentially be used to release oxygen to improve wound healing in the absence of being bound to a wound dressing, and/or in the absence of a biological scaffold. In this embodiment, after application of the oxygen-loaded zeolites to the wound environment, it may be desirable to cover the wound environment to retain the released oxygen in the wound environment.

[00106] In certain embodiments, an oxygen-loaded zeolite biological scaffold may be produced in a similar manner to the methods described herein, but without applying the scaffold to the wound dressing precursor layer. Such a scaffold may optionally be used in conjunction with a wound dressing (i.e., without being bound to the dressing) as it may be desirable to cover the wound environment to retain the release oxygen in the wound environment.

[00107] Accordingly, in a further aspect, the present disclosure provides a use of oxygen-loaded zeolites, wherein the oxygen-loaded zeolite releases oxygen to improve wound healing.

[00108] In certain embodiments, the oxygen-loaded zeolites are prepared by

(b) lyophilising the oxygen-binding zeolite suspension; and

[00109] In certain embodiments, step (a) further comprises mixing the oxygen-binding suspension with a suitable provided biocompatible polymer that provides a biological scaffold to the oxygen-binding zeolite suspension. In certain embodiments, step (a) further comprises applying the oxygen-binding zeolite biopolymer mixture to a dressing layer or a mould to form an oxygen-binding zeolite biopolymer layer. Suitable polymers are discussed herein.

[001 10] In certain embodiments, the oxygen-loaded zeolites are prepared by

(b) providing a biopolymer mixture; (c) mixing the oxygen-binding zeolite suspension with a biopolymer mixture to produce an oxygen-binding zeolite biopolymer mixture;

(e) lyophilising the oxygen-binding zeolite biopolymer mixture; and

(f) exposing the oxygen-binding zeolite biopolymer mixture to oxygen to form oxygen- loaded zeolites.

[001 1 1 ] In certain embodiments, the biopolymer is not gelatin. In certain embodiments, the zeolite is not copper-activated faujasites.

[001 12] The invention is hereinafter described by way of the following non-limiting examples and accompanying figures.

EXAMPLES

Example 1 - Fabrication and characterisation of oxygen releasing wound dressings

1001 13] Fabricating zeolite dressings

[001 14] 1 % w/v of agarose (Bio-Rad Certified Molecular Biology Agarose) was mixed with 1 % w/v of alginate (Sigma Aldrich). Alginate/agarose serves as a biocompatible matrix to uniformly disperse the active component, (ie zeolites). Both of these biopolymers are medically certified polymers used for wound management.

[001 15] Method 1 : Zeolite- 13X with a diameter of 1-2 micron was obtained from Sigma Aldrich.

Zeolite- 13X was chosen as a result of its high oxygen adsorption and capacity. Different quantities of zeolite-13X (0.5, 2.5, 5, 10 and 20 g) were weighed separately, dispersed in water ( 10 mL) and sonicated using a GT SONIC® Ultrasonic cleaner (at a frequency of 333/40 kHz, ultrasonic power 120W and heating power 100W) for 10 minutes. The sonicated zeolite suspensions were added slowly using a syringe into 40 mL of the alginate/agarose mixture under constant stirring using a magnetic stirrer to ensure uniform distribution of zeolites throughout the mixture (reducing the chance of agglomeration), with final zeolite concentrations of 1 , 5, 10, 20 and 40 % w/v. Medical grade gauzes were placed at the bottom of 24, 48 or 96 well plates. The zeolite biopolymer solution was accurately transferred into the well plates using a micropipette and stored at -80 °C. The frozen samples were then lyophilised for 48 h to provide circular wound dressings. The lyophilisation removes most of the water and trapped gases. The lyophilised dressings with zeolite loadings 0.5, 2.5, 5, 10 and 20 g/g zeolite/biopolymer were exposed to the atmosphere to backfill the zeolite pores with atmospheric gases, including oxygen.

[001 16] Method 2: Zeolite- 13X with a diameter of 1-2 micron was obtained from Sigma Aldrich.

Zeolite- 13X was chosen as a result of its high oxygen adsorption and capacity. Different quantities of zeolite-13X (0.5, 2.5, 5, 10 and 20 g) were weighed separately, dispersed in water (10 mL) and sonicated using a GT SONIC® Ultrasonic cleaner (at a frequency of 333/40 kHz, ultrasonic power 120W and heating power 100W) for 10 minutes. The sonicated zeolite suspensions were added slowly using a syringe into 40 mL of the alginate/agarose mixture under constant stirring using a magnetic stirrer to ensure uniform distribution of zeolites throughout the mixture (reducing the chance of agglomeration), with final zeolite concentrations of 1 , 5, 10, 20 and 40 % w/v. The zeolite biopolymer solution was accurately transferred into 24, 48 or 96 well plates using a micropipette and stored at -80 °C. The frozen samples were then lyophilised for 48 h to provide circular wound dressings. The lyophilisation removes most of the water and trapped gases. The lyophilised samples with zeolite loadings 0.5, 2.5, 5, 10 and 20 g/g zeolite/biopolymer were placed in an oven under vacuum for 2 hours, backfilled with pure oxygen for 15 minutes, and then rapidly sealed in an oxygen environment.

[001 17] The control dressing without zeolites was labelled as AA. Dressings with zeolite concentrations of 0.5, 5, 10 and 20 g/g zeolite/biopolymer were labelled as AAZ1, AAZ2, AAZ3 and AAZ4, respectively. The concentrations of zeolites at various stages of preparation are shown in Table 1.

[001 18] Table 1 : Concentration of Zeolite 13X in prepared samples

[001 19] Morphological characterisation using SEM analysis [00120] The surface morphology of the lyophilised (dry state) dressings and zeolite powder was evaluated by excising thin sections, followed by examination via scanning electron microscopy (Crossbeam 540 SEM ; Zeiss, Germany), using a 30 kV beam of secondary electrons under high vacuum (6x 10^"4 Pa). Samples of lyophilised dressings were prepared by taking a thin section of the dressing using a razor blade. The section was then directly examined under SEM. Zeolite samples were prepared by adding droplets of well dispersed zeolites on aluminium stub and further dried.

[00121 ] SEM images revealed that the pores of the alginate/agarose scaffold of AAZ1 , AAZ2 and

AAZ3 differ greatly in pore size distributions (Figure la, lb and lc, respectively). There were less pores in AAZ3 compared to AAZl and AAZ2. This shows that a higher concentration of zeolites affects the formation of highly porous architectures, with a higher concentration of zeolites resulting in a structure with a lower number of larger pores. The majority of pores in AAZl and AAZ2 have a diameter that lies in the range of 50-100 μιη and 200-250 μιη in size, respectively. 27.27% of pores in AAZ3 are in the range of 100-150 μτη, 21.21% in the range of 150-200 μιη and 18.18% are in the range of 200-250 μιη. In other words, AAZ3 has less pores that are typically larger in size compared to AAZ l or AAZ2.

[00122] Figure 2 shows the AAZ2 dressing at l OOx magnification, with the arrows indicating the location of zeolite particles (approximately 2 μηι in size) distributed in the dressing.

[ 00123] Figure 3 provides SEM images of (a) zeolites alone at high magnification (Scale bar: 20 μιη, magnification: 1000 x); and zeolites impregnated on the surface of AAZ3 at (b) low magnification (Scale bar: 100 μιη, magnification: 200 x) and (c) high magnification (Scale bar: 20 μιη, magnification: 1000 x);

[00124] Mechanical properties

[ 00125] The mechanical properties of the dressings were analysed using a universal testing instrument (EZ test, Shimadzu). Compression testing was conducted on cylindrical specimens ( 1 1 mm diameter, 5 mm height) placed between metal plates and using a 50 N load cell, in dry states (Figure 4 (a)) as well as swollen states (Figure 4(b)). Triplicates of samples were tested and their averages were reported including standard deviation (SD).

[00126] Trends in the mechanical properties of the dressings were opposite in the dry and wet states. AA showed the highest modulus in the dried state, whereas incorporation of zeolites led to a decrease in modulus value. In swollen states, dressings gained mechanical strength on addition of zeolites. The compression modulus was found to increase with an increase in concentration of zeolites/polymer up to 10 g/g. At 20 g/g of zeolites/polymer, the modulus of the dressings decreases, presumably due to poor distribution and agglomeration of the zeolite particles. While not wanting to be bound by theory, this may be because the zeolites exhibit hydrophilic behaviour when water molecules interact with their surface. Zeolites play the role of fillers in a composite mixture and the hydrophilic nature of the zeolites enable the fillers to better interact with the polymers in the swollen state and show increased mechanical strength.

[00127] Swelling experiments

[00128] The ability of a dressing to swell is of interest, as dressings that have a good ability to swell may allow better absorption of wound exudate. The water uptake (swelling) of the dressings was measured over 15 days. Equal weights of dressings were immersed in deionized water at 37 °C. The soaked dressings were removed from the water after periods of time, blotted on filter paper to remove surface water and weighed. The initial and final weights were noted as W l and W2, and the percentage of water uptake was determined according to equation 2, and expressed as mean ± SD (n = 3):

Percentage of swelling = [(W2-W 1 )/W 1 ] ^χ 100% (2) where, Wl and W2 are the initial and final weights, respectively.

[00129] After 1 day, the swelling index was found to increase with increasing zeolite/polymer concentration up to 5 g/g (Figure 5), which is consistent with increased water uptake into the dressings and zeolite pores. After 10 days, equilibrium is reached for all dressings at a swelling index of approximately 40.

[00130] Oxygen release studies under hypoxic conditions

[0013 1 ] MilliQ water was deoxygenated in a container fitted with an oxygen probe and then the test materials were added under a blanket of nitrogen and the container was sealed, the test materials being (a) mock control with no addition, (b) dressing with Og/g zeolite/biopolymer (AA), (c) dressing with 0.5g/g zeolite/biopolymer (AAZ l ), or (d) equivalent amount of zeolites alone as present in the dressing with 0.5g/g zeolite/biopolymer, and the system was sealed and dissolved oxygen release was monitored. From the oxygen release data obtained (Figure 6), the dressing without zeolites (AA) released oxygen initially due to gases trapped within pores of the dressing and then displayed a linear increase consistent with the control (mock addition). In comparison, wound dressings with 0.5g/g zeolite/biopolymer (AAZl ) resulted in greater initial release of oxygen over 3 h and then a sustained increase up to approximately 20 h. Zeolites alone resulted in a more rapid release of oxygen and saturation of the solution after approximately 16 h. This result shows that the wound dressing impregnated with zeolites can bind oxygen and release the oxygen when in solution. [00132] The same system was also used to monitor the release of oxygen from dressings with different concentrations of zeolites (Figure 7). An increase in zeolite loading led to an increase in oxygen release into the system. In particular, the dressing containing 0.5g/g zeolite/biopolymer had released approximately 6 ppm dissolved oxygen after 15 h; whereas the dressing containing 5g/g zeolite/biopolymer had released approximately 8 ppm dissolved oxygen after 15 h. The dressing containing lOg/g zeolite/biopolymer had released approximately 17 ppm dissolved oxygen after approximately 14 h.

[00133] Cell culture experiments

[ 00134] Hoechst staining - For the assay, 3T3 mouse fibroblast cells were seeded onto 12 well plate at a density of l x 1()⁴ cells/well and incubated under standard culturing conditions. Extracts from the dressings were prepared by incubating the pre-sterilized dressings containing either Og/g or 0.5g/g zeolite/biopolymer incubated in culture medium for 24 h and 48 h at 37 °C and the medium containing leachables from the dressings was collected. Culture media of the seeded cells was replaced after 24 h by the extract (media with the leachables). Cells were incubated with the extract for 24 h. After the incubation period, the extract was replaced with fresh media containing 2μg/ml of Hoechst and incubated for 10 minutes, away from light. The staining solution was removed and the cells were washed 3 times in PBS and viewed under Nikon eclipse fluorescent microscope.

[00135] As shown in Figure 8, there was higher cell density for cells exposed to the AAZ1 dressing extract, relative to that of cells not exposed to extracts (control). Cell density was less in cells exposed to AA. These results indicate that the AAZ dressing results in higher cell proliferation due to increased oxygen supply by zeolites impregnated within dressings.

[00136] FDA/PI staining

[00137] Live and dead 3T3 cells after treatment with AAZl (with 0.5g/g zeolite/biopolymer) extract prepared as above was determined by staining with fluorescein diacetate (FDA) and propidium iodide (PI). Around 5 ^χ 10⁴ cells were seeded onto each well of 24 well plates, followed by 24 h incubation in dressing conditioned medium of AAZl at 37°C in 5 % C0₂ incubator. After incubation, the medium was removed and cells were washed with IX phosphate buffered saline (PBS). They were then incubated in staining solution (5 mg/ml of FDA and 2 mg/ml of PI in DMEM medium) for 5 minutes and washed with IX PBS. Then cells were visualised under a Nikon Eclipse fluorescent microscope.

[00138] Cell viability was assessed using live/dead FDA/PI staining to visualise whether cells are viable when they are incubated in the presence of the dressing extracts (Figure 9). Positive controls (dead cells) were cells treated with Triton X (no dressing) and negative controls (live cells) were cells incubated in cell culture medium alone (no dressing). The cells incubated in the presence of the extract of the dressing containing 0.5g/g zeolite/biopolymer were viable, with similar cell viability as the negative control cells. This shows that the extract from the dressing is not detrimental to cell growth.

[00139] MTT assay

[00140] The viability of cells seeded on the AA, AAZl , AAZ2, and AAZ3 scaffolds were evaluated using the colorimetric MTT (3-(4, 5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT assay evaluates the reduction of the tetrazolium component MTT by viable cells. Therefore, the level of the reduction of MTT into formazan can determine the level of cell metabolism. For the assay, cells were seeded onto a 96 well plate at a density of lxl 0⁴ cells/well and incubated under standard culturing conditions. Extract from the scaffolds were prepared by incubating the pre-sterilized scaffolds in culture medium for 24 h and 48 h at 37 °C and the medium with leachables was collected. Culture media of the seeded cells were replaced after 24 h by the extract (media with the leachables). Cells were incubated with the extract for 24 h. After the incubation period, the extract was replaced with fresh media containing 10% MTT solution. The plates were then incubated at 37 °C in a humidified atmosphere. After 4 h of incubation, the medium was removed and 100 μΐ of solubilisation buffer was added to each well to dissolve the formazan crystals. The absorbance of the lysate was measured in a micro plate reader (Biotek) at a wavelength of 570 nm. Leachables from scaffolds incubated in culture medium were used as positive control.

[00141 ] The metabolic activity of 3T3 fibroblast cells in the presence of various loadings of zeolite-13X alone at 100, 50, 25, 12.5, 6.25, 3.125, 1.56 and 0.78 mg/ml of Zeolite 13X (alone) was measured using the MTT assay (Figure 10). Positive control refers to cells treated with Triton X (dead cells) and negative control refers to cells incubated in cell culture medium alone (live cells). As shown in Figure 10, cell viability was close to that of the negative control for zeolite concentrations of less than 12.5 mg/ml, and concentrations of 25 and 12.5 mg/ml having in excess of 90% viability.

[00142] The metabolic activity of 3T3 fibroblast cells in the presence of extracts of dressing with

0, 0.5, 10 or 20 g/g zeolite/biopolymer was measured using the MTT assay (Figure 1 1 ). The dressings were incubated in medium for 24 h and then the cells were incubated with this conditioned medium for 24 h before the assay. In all cases, the dressing extracts were deemed to be non-cytotoxic.

[00143] Cell culture experiments under hypoxic conditions

[00144] 3T3 mouse fibroblasts, Hacat cells, and human dermal fibroblasts cell lines were used in these studies. Initially, 3T3 mouse fibroblast cells were examined under nonnoxia and hypoxia conditions using FDA staining and fluorescent microscopy. Normoxia treated cells were cultured at 37 °C for 24 h with 21 % oxygen (negative control; Figure 12; normal cells). Hypoxia treated cells were cultured at 37 °C for 24 h with 0. 1% oxygen (positive control; Figure 12; cells under hypoxia). Subsequently, these cells were cultured for another 24 h under hypoxia with either fresh media or fresh media containing dressings with 0.5g/g zeolite/biopolymer . Images revealed that there was a drastic decrease in cell number when 3T3 cells were cultured in hypoxic conditions (at 0.1% oxygen) versus the control (at 21% oxygen) (Figure 12). However, cell number increased significantly when the media was replaced with fresh media containing extracts from the dressing with zeolite and mcubated for 24 h versus the control (medium without dressing).

[00145] Further evaluation of the dressing containing 0.5g/g zeolite/biopolymer was conducted using human dermal fibroblasts cells (primary cells) and phase contrast and fluorescent imaging. Cells were incubated under normoxia (21% oxygen) for 24 h or hypoxia (0.1 % oxygen) for 48 h (compared to normoxic conditions, cells take more time to proliferate in hypoxic conditions). The cells cultured under hypoxia were then treated with fresh media containing the extract of the dressing with 0.5g/g zeolite/biopolymer as described above and cultured for another 24 h under hypoxic conditions. As shown in Figure 13, the cells were swollen and blebbing after 48 h under hypoxic conditions. While not wanting to be bound by theory, it is thought that when hypoxic cells are devoid of oxygen, cells are unable to produce sufficient ATP for the sodium channels to control the level of sodium within the cells. As a result, there is uncontrolled uptake of water and sodium which results in cell swelling and blebbing. The endoplasmic reticulum becomes swollen and releases the proteins attached on the ribosomes. Cells undergo anaerobic glycolysis by which more lactic acid is produced within the cells which can denature proteins. This change appears to be reversed when cells under hypoxia are exposed to the extracts from oxygen releasing dressings.

[00146 ] Wound scratch assays

[00147] The scratch assay is a well-established method to measure cell migration in vitro. The basic steps include creating a "scratch" in a confluent cell monolayer, capturing the images at the beginning and at regular intervals during cell migration to close the scratch, and comparing the images to study the migration rate of the cells.

[00148] For the wound scratch assay using Hacat cells under normoxia, the cells were seeded at a density of 2* 10⁵ cells/well and incubated at 21% oxygen for 24 h. A scratch was made using a 1 mL sterile pipette tip and the debris was removed by washing with PBS. Subsequently, media (control) or conditioned media (in which dressings with 0.5g/g zeolite/biopolymer were equilibrated for 24 h) was added and the cells were incubated at 21% oxygen and 37°C. Images were taken after 0, 24 and 48 h. Under normoxic conditions, the wound scratch assay demonstrated that Hacat cells migrated and proliferated more rapidly to cover the scratched area when they were exposed to dressing conditioned media versus the control (Figure 14), demonstrating increased cell motility in case of cells incubated in conditioned media.

[00149] For the wound scratch assay under hypoxic conditions, human dermal fibroblasts

(passage 5) were seeded into well plates at a density of 2x 10^s cells/well, and incubated at 0. 1 % oxygen for 48 h so they reached 80% confluency. A scratch was made using a 1 mL sterile pipette tip and the debris was removed by washing with PBS. Subsequently, media (control) or conditioned media (in which dressings with 0.5g/g zeolite/biopolymer were equilibrated for 24 h) was added and the cells were incubated at 0.1% oxygen and 37°C. Images were taken after 0, 24 and 48 h. Human dermal fibroblast cells proliferated well when they were exposed to media containing dressing (Figure 15) demonstrating increased cell motility in case of cells incubated with dressing extract.

[00150] Stability testing- Physical and Mechanical Properties

100151 ] Stability testing determines the effect of environmental factors on the quality of the wound dressing, along with the testing performed to evaluate functionality of the dressing including mechanical performance and ability to release oxygen. Lyophilisation allows long term storage of samples without any appreciable differences in properties. The physical and mechanical properties of samples prepared at different time points (Batch 1 , Batch 2 and Batch 3) were tested and stored at room temperature (Table 2). We observed that there was no significant change in the properties of lyophilised samples namely, Batch 1 , Batch 2 and Batch 3 with respect to physical appearance and mechanical performance.

[00152] Table 2: Stability analysis- Physical and Mechanical Parameters of different batches of samples

[00153] Stability testing- Dissolved Oxygen

[00154] Dissolved oxygen release studies of samples prepared at different periods and stored at room temperature showed that there was no significant change in the release of oxygen from all samples. Samples prepared before 6 months and 3 months showed slightly higher release of oxygen than freshly prepared samples. This might be because of the adsorption of more oxygen by zeolites during the passage of time (Figure 16).

[00155] In vivo studies

[00156] A partial thickness wound was created in Balb/c mice using a 70 mm² total body surface area scald wound. This model has been approved and used previously in wound healing and scarring (Adams et ai, 2009). This established murine model can be used to study the efficacy of wound dressings and their ability to release oxygen and thereby accelerate wound healing.

100157] The study was conducted using three different would dressings:

a. Zeolite impregnated agarose/alginate dressings, i.e. the wound dressing of the present disclosure (designated 'T' for test dressing);

b. Agarose/alginate dressings, i.e. the wound dressing of the present disclosure without the oxygen-releasing component (designated 'CI ' for control dressing 1 ), and;

c. A commercially available burn wound dressing - Algisite™ (Smith and Nephew)

(designated 'C2' for control dressing 2).

[00158] Two controls were selected in this study to compare the performance of the wound dressing of the present disclosure with an existing commercial product ( C2), and to show that it is the oxygen- releasing characteristics that are responsible for the observed effect (and not the biopolymer component - C I).

[00159] End-points of 4 days and 12 days were selected so that the wound healing process and rate of healing with different dressings could be studied by histology at shorter time points.

[00160] In all of the results shown below, the following abbreviations represent:

T - Zeolite impregnated wound dressing (test material)

C 1 - Control dressing without zeolite

C2 - Algisite™ (Smith and Nephew dressing).

[00161 ] In each dressing group there were 4 mice (n = 4) that did not remove their dressings during the experiment: only results from these mice are compared.

[00162] Visual analysis of the wound healing process

[00163 ] Macroscopic and microscopic images of the wounds at 0, 2, 4, 6, 8, 10 and 12 days for each of the wound dressings and replicates (n = 4) were obtained. [00164] The microscopic images showed that the size of the wound had reduced to a greater extent for wounds treated with T as compared to those treated with C I and C2. Analysis of the wound area and determination of the percentage reduction (Figure 17) in size at day 12 revealed a statically significant difference for wounds treated with T as compared to C I and C2.

[00165] Ultrasound imaging of the wound area at day 12

[00166] Ultrasound imaging was used to image the epidermis and dermis layers of the wounds in mice (n = 4) treated with the different dressings and compared to the skin of a healthy mouse. In general, a more intact epidermis and dermis was noted for wounds threated with T, indicating that the epidermis has regrown back to a greater extent with a faster rate of re-epithelialisation.

[00167] Skin multi-parameter analysis

[00168] Skin elasticity measured on day 12 was on average higher for C I and T compared to C2, although not statistically significant. Transepidermal water loss (TEWL), a marker of cutaneous barrier function is supposed to be high at the time of wound induction, whereas it decreases thereafter. This trend appears to be observed for all of the dressings, although a more rapid decrease and return to baseline (normal skin value) is noted for dressing T. Healthy skin should have a pH of 6.4, whereas chronic wounds tend to have an alkaline pH. As the wound heals we expect to see a gradual decrease in pH. For T, the average pH was acidic from day 6 onwards, whereas the pH for CI and C2 was neutral or slightly alkaline. In the case of wound healing, skin hydration is initially high, and should return back to a baseline value upon healing. This trend was noted for all of the dressings with no significant differences. Skin colour (based on melanin concentration) was observed to increase for all of the dressings which is consistent with the formation of a scab over the wound.

[00169] Immunohistochemistty

[00170] Figure 18 demonstrates that there is higher infiltration of macrophages for wounds treated with C2 and C 1 when compared to T. This indicates the ability of T to reduce inflammation to a greater extent.

[00171 ] Histology

[00172] Figure 19 demonstrates that after 12 days much greater collagen deposition is noted with wounds treated with T as compared to CI and C2, which is consist with faster re-epithelialisation [00173] Throughout the specification and the claims that follow, unless the context requires otherwise, the words "comprise" and "include" and variations such as "comprising" and "including" will be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[00174] The reference to any prior art in this specification is not, and should not be taken as, an acknowledgement of any form of suggestion that such prior art forms part of the common general knowledge.

[00175] It will be appreciated by those skilled in the art that the invention is not restricted in its use to the particular application described. Neither is the present invention restricted in its preferred embodiment with regard to the particular elements and/or features described or depicted herein. It will be appreciated that the invention is not limited to the embodiment or embodiments disclosed, but is capable of numerous rearrangements, modifications and substitutions without departing from the scope of the invention as set forth and defined by the following claims.

REFERENCES

[00176] Adams DH, Ruzehaji N, Strudwick XL, Greenwood JE, Campbell HD, Arkell R, Cowin

AJ. Br J Dermatol. 2009 Aug; 161 (2):326-36.

[00177] Sheffield PJ. Tissue oxygen measurements. In: Problem Wounds: the Role of Oxygen

(Davis JC, Hunt TK, eds). New York: Elsevier, 1988; 17-52.

Claims

1. A wound dressing comprising an oxygen-loaded zeolite, wherein the oxygen-loaded zeolite releases oxygen when in use.

2. The wound dressing of claim 1 , wherein the oxygen-loaded zeolite is incorporated within a biopolymer matrix.

3. The wound dressing of claim 1 , wherein the oxygen-loaded zeolite is incorporated within a biopolymer matrix comprising alginate and agarose.

4. The wound dressing of any preceding claim, wherein the oxygen-loaded zeolite releases dissolved oxygen when the wound dressing is applied to a wound.

5. The wound dressing of any preceding claim, wherein the released oxygen improves wound healing.

6. The wound dressing of any preceding claim, wherein the wound dressing comprising an oxygen- loaded zeolite comprises a layer of oxygen-loaded zeolites incorporated within a biopolymer scaffold, wherein the layer has been lyophilised and then loaded with oxygen.

7. The wound dressing of any preceding claim, wherein the oxygen-loaded zeolite is selected from the group consisting of Zeolite 13X, fluorinated Zeolite Y, NaX zeolite, NaY zeolite, faujasites, Zeolite Socony Mobil-5 (ZSM-5), MFI type zeolites, and mordenites.

8. The wound dressing of any preceding claim, wherein the oxygen-loaded zeolite is Zeolite 13X.

9. The wound dressing of any preceding claim, wherein the wound dressing is in a form selected from a hydrogel dressing, a foam dressing, hydrocolloid dressing, absorbent dressing, gelling fibre dressing, hydroselective dressing or an alginate dressing.

10. The wound dressing of any preceding claim, wherein the wound is selected from a burn, a chronic wound, a hypoxic wound, a venous leg ulcer, a diabetic foot ulcers, a laceration, and an incision.

1 1. The wound dressing of any preceding claim, wherein the wound is hypoxic.

12. The wound dressing of any preceding claim, wherein the zeolite is provided within the dressing at a concentration between 0.001 mg/cm³ and 1000 mg/cm³.

13. The wound dressing of any preceding claim, wherein altering the concentration of the oxygen -binding zeolite within the wound dressing correlates with the amount of oxygen released.

14. A method of producing an oxygen-loaded zeolite layer for the wound dressing of any one of the preceding claims, the method comprising:

(b) providing a biopolymer mixture;

15. The method of claim 14 wherein step (e) comprises freezing the oxygen-binding zeolite biopolymer layer and then lyophilising the oxygen-binding zeolite biopolymer layer.

16. The method of claim 14 or 15, wherein step (d) comprises applying the oxygen-binding zeolite biopolymer mixture to a structural layer within a mould to fonn an oxygen-binding zeolite biopolymer layer.

17. Use of the wound dressing comprising an oxygen-loaded zeolite of any one of claims 1 to 13 or the use of the oxygen-loaded zeolite layer for the wound dressing produced by the method of claims 14 or 15.

18. Use of claim 16 to improve wound healing.

19. Use of an oxygen-loaded zeolite, wherein when in use, the oxygen-loaded zeolite releases oxygen to improve wound healing.

20. Use of claim 19, wherein the oxygen-loaded zeolite is prepared by (a) dispersing a predetermined amount of an oxygen-binding zeolite in a solution to produce an oxygen-binding zeolite suspension;

(b) lyophilising the oxygen-binding zeolite suspension; and

21. Use of claim 20 wherein step (a) further comprises adding a biopolymer that provides a biological scaffold to the oxygen-binding zeolite suspension.

22. Use of any one of claims 16 to 20, wherein the wound is hypoxic.