WO2021015631A9 - Multilayer nanofibres with bioactive compositions for wound healing management - Google Patents

Abstract

Described herein are multilayer nanofibre compositions with bioactives for wound healing management and related methods. More specifically, a matrix comprising a composition of multifunctional nanofibres layered together including at least one or more functional bioactives in each layer to separately or work together synergistically for the management of the full wound cycle thereon. Upon exposure to moisture or on wet skin, each layer of the nanofibre composition dissolves at a desired controlled rate to release the bioactive or a non-dissolvable layer may function as a sacrificial skin layer.

Description

MULTILAYER NANOFIBRES WITH BIOACTIVE COMPOSITIONS FOR WOUND HEALING

MANAGEMENT

TECHNICAL FIELD

Described herein are multilayer nanofibre compositions with bioactives for wound healing management and related methods. More specifically, a matrix comprising a composition of multifunctional nanofibres layered together, including at least one or more functional bioactives in each layer to separately or work together synergistically for the management of the full wound cycle thereon.

BACKGROUND ART

Delivery of bioactives with nanofibre compositions is an issue spanning many prior art documents. A common theme is the incorporation of bioactive or bioactives into or onto a substrate that may be used to assist delivery, for example, to the skin of a patient.

The use of electrospinning to produce a nanofibre matrix that contains an active is known. See for example US Patent No. 7,732,427 and WO 2013/035072. However, there remains a need for active agent delivery systems that are particularly suitable for wound management and delivery to mammalian skin. One problem with prior art nanofibre and active agent combinations is that the actives are not controllably released to the target area(s).

Wound management has remained a challenging clinical process for decades. The wound healing cycle involves four phases: haemostasis, inflammation, proliferation and remodelling and each phase assisted by enzymes, proteins, growth factors, and nutrients (see Figure 1). There have been numerous resources directed towards wound care management with an emphasis placed on new therapeutic approaches. The wound healing process is a continuous and coordinated process that involves multiple cell populations, collagen remodelling, the extracellular matrix, and the reaction of growth factors and cytokines.

Even though all wound types go through a similar process, the time frame varies based on the types of wounds. Wounds are clinically categorized as being acute, chronic, and complicated based on the complexity and the healing time. Therefore, for a wound dressing product to act to its full benefit, it should deliver drugs (if it is intended to deliver drugs) faster, but also for a longer period for quick recovery.

Commercial wound dressing products currently utilize different woven and synthetic materials with generic drugs to support the wound healing process. In particular, collagen as freeze-dried sheets, pads, pastes, and gels have traditionally been well received as a wound dressing material as it stimulates new tissue growth by attracting cells, such as fibroblasts and keratinocytes to the wound.

Fibroblasts secrete a variety of growth factors (GF) such as such as epidermal GF (EGF), insulin-like GF (IGF-1), platelet-derived GF (PDGF), and transforming GF-beta (TGF-b), which guide the formation of Extra Cellular Matrix (ECM) which in turn regulates the cellular function to grow new skin. When an excess of collagen peptides is supplied through the dressing, it promotes ECM formation to achieve granulation and proliferation.

Also, keratinocytes secrete a variety of GFs and cytokines such as IL-1, TGF-b. When fibroblasts and keratinocytes migrate across the wound, different processes such as re-epithelization and remodelling is achieved. Again, this process is shown in Figure 1.

Traditional wound care products are designed to provide the wound's macro-environment protection, including moist wound environment control, fluid management, and controlled transpiration of wound fluids. The newer classes of wound healing biomaterials (mostly collagen-based) are also designed to target specific defects, stimulate and recruit particular cells in the chronic wound environment.

These new classes of commercial collagen-based wound dressing materials available in the market employ a variety of agents such as EDTA to deactivate proteases, oxidized regenerated cellulose (ORCs), antimicrobial agents, and growth factors to accelerate the healing process. The collagen from these products is derived from a bovine, porcine, equine, or avian source, which is purified to render non- antigenic properties. The collagen is non-dissolvable and designed to absorb excess fluid during dressing. They are either 'Type I' (native triple helix) or denatured (gelatin) and available in various pore sizes and surface areas. They can come in the form of fibres, freeze-dried "pad" or gel film. The type of collagen, pore size, and surface area, are the main attributes considered to enhance wound management. Studies have shown, Type I native collagen can attract MMP-1, and denatured gelatin attracts MMP-2, MMP-9, stromelysin, and matrilysin. These MMPs (among others) are found in excess in chronic wounds and contribute to a wound's chronicity. Research has also shown that the hydrophilic property of the dressing material can significantly influence fibroblast formation.

As above, current collagen-based wound dressing products are sourced from a bovine, porcine, and equine. For example, BIOSTEP™ (Smith & Nephew) combines porcine-derived type I collagen and gelatin with EDTA, alginate, and carboxymethyl cellulose (CMC) to promote the wound healing process. Flowever, this product requires additional additives to work on wound exudate.

BIOPAD™ (Argentum Medical) is an equine collagen pad to control minor bleeding and is also claimed to promote cell migration and inhibition of MMPs. Flowever, the BIOPAD™ is a collagen film that transforms into a gel upon application. There are other wound healing products such as COLACTIVE™ (Smith & Nephew), COLLI EVA Ag™ and DermaSIL™ (CollMED Lab), DermADAPT™ (Pegasus Biologies), FIBRACOL PLUS™ (Johnson & Johnson), and OASIS™ (Healthpoint) that all work on similar principles, but are manufactured from bovine, porcine and equine sources and are sold in the form of gel, powder, sheet or films.

The wound healing products above are based on collagen; they are derived from equine, bovine, and porcine sources. The outbreaks of various diseases among these animals have strengthened regulatory approvals, and religious constraints have limited the use and appeal of equine, bovine, and porcine collagen in bio-medical skin and oral delivery applications.

US Patent No. 9,775,917 discloses a method of delivering bioactives with dissolvable collagen nanofibre. However, this composition is not suitable for wound management as the wound exudate and haemostasis part of wound healing management requires a nanofibre to have the physical strength to substitute a bandage and allow a controlled rate of release and penetration of actives to work on MMPs inhibition.

US Patent No. 2013/0018336 relates to a repositionable bandage including an adhesive hydrocolloid mass with microfibre and nanofibre web coating. This invention uses nanofibre to address technical problems of painlessly re-positioning hydrocolloid mass structures by modifying the surface with a web of dissolvable or gelling nanofibre in contact with the wound. However, a disadvantage of this bandage is that it requires a hydrocolloid mass surface to act upon haemostasis.

CN 103990175B discloses a double-layer nanofibre with antibiotics for a wound dressing. Here one layer is water-soluble, and the other layer is oil soluble carrying drugs with different molar mass. However, a disadvantage of this wound dressing is that the rate of release of the drugs is administrated by controlling or adjusting the molar mass of the antibiotics, hence limiting the variety of drugs that can be used with this dressing.

From the above, it would be useful to have a one-step multi-layered marine collagen product proven for use in the wound care in the market with minimal dressing routines and minimum possibilities of any transmitted diseases and a structure aligned to the natural triple helix collagen structure found in the human skin or at least to provide the public with a useful choice of source.

Further aspects and advantages of the nanofibre and bioactive compositions and their usage will become apparent from the ensuing description that is given by way of example only.

SUMMARY

Described herein is a multilayer nanofibre polymer composition comprising non-woven mesh, including one or more bioactive compounds. In particular, the mesh comprises one or more polymer nanofibres with different properties. The therapeutic bioactives may be chemically or physically bonded with the nanofibre matrix. Upon exposure to wet or moist skin, one or more of the nanofibres may dissolve rapidly, thereby releasing actives, while the other layer (s) of nanofibre polymers may remain and degrade over a further period thereby releasing further actives.

In a first aspect there is provided a multilayer matrix nanofibre composition for wound healing management comprising: at least two nanofibre layers; and an effective amount of at least one bioactive compound physically and/or chemically bonded within the nanofibre layer matrix; the bioactives in at least one layer are segregated from bioactives in at least one other layer preventing any reactions between the bioactives in each layer during storage of the composition under ambient conditions substantially free of moisture; wherein the first layer nanofiber is formed from a base material that is solubilised with the bioactives and electrospun together or the bioactives are coated on the nanofibre surface via spray coating or dip coating; wherein the second layer nanofibre is formed from a base material that is solubilised with the bioactive or bioactives in an aqueous-based solvent solution and the base material and bioactives are together spun via electrospinning to form dry fibres; and wherein on exposure to moisture, at least one of the nanofibre layer dissolves and/or degrades to slowly to release actives over a period of time and/or to act as a sacrificial protective skin layer, and the other nanofibre layer(s) dissolves rapidly, thereby substantially releasing all of the at least one bioactive compound.

In a second aspect, there is provided a method of treating a wound by applying at least one bioactive compound to a subject to treat and penetrate the skin of the subject, the method comprising the steps of:

(a) selecting a multilayer matrix nanofibre composition substantially as described above;

(b) moistening the composition; and

(c) applying the composition as a wound dressing to the skin of the subject.

In a third aspect, there are provided methods of skin treatment of a subject in need thereof by application of the composition by a skin dressing substantially as described above, the treatments selected from promoting skin care or repair; assisting or enhancing wound healing; addressing a microbial infection; treating or preventing inflammation; promoting or enhancing cell proliferation; preserving or improving skin elasticity; preserving or improving skin moisture retention; delivering at least one antioxidant to skin; improving scars; and combinations thereof.

In a fourth aspect, there is provided the use of a composition substantially as described above, in the manufacture of a medicament for treatments selected from promoting skin care or repair; assisting or enhancing wound healing; addressing a microbial infection; treating or preventing inflammation; promoting or enhancing cell proliferation; preserving or improving skin elasticity; preserving or improving skin moisture retention; delivering at least one antioxidant to skin; and combinations thereof.

In a fifth aspect, there is provided a skincare product comprising the composition substantially as described above, wherein the product is in the form of: a plaster, a bandage, patch, mask and a dressing, In a sixth aspect, there is provided a method of producing a composition substantially as described above by the steps of:

(a) selecting at least one bioactive compound;

(b) selecting at least one nanofibre base material;

(c) mixing the bioactive compound or compounds and the nanofibre base material or materials with water or acid to form an aqueous solution; and

(d) electrospinning the solution to form one or more nanofibres physically and/or chemically bonded with the bioactive compound(s).

In a seventh aspect, there is provided a method of producing a multilayer matrix substantially as described above by the steps of:

(a) producing a first layer by the steps of:

I. providing an active agent or agents;

II. providing a nanofibre base material;

III. mixing the active agent or agents and the nanofibre base material in a solvent phase to form a solution;

IV. electrospinning the solution to form one or more nanofibres chemically bonded with the bioactive(s) in a first layer;

V. electrospinning the base material into nanofibre;

VI. coating the nanofibre with active agents using spraying coating or dip coating method; and

VII. electrospinning the active agents directly with the polymer solution to form active embedded nanofibre matrix.

(b) forming a second or further layer by separately repeating steps (I) to (IV) to form an additional layer; or

(c) combining the first and subsequent layers to form a multilayer composition.

In an eight aspect, the described multilayer matrix nanofibre composition may be altered to contain two layers to three or more layers depending on the wound application. For example, for the acne scarring application the design may consist of just two nanofibre layers; one to provide moisture benefit whereas other layer may dissolve into the skin to provide collagen and carried drugs. Whereas for surgical wounds, the design may comprise three to four layers to provide additional benefits for haemostasis in addition to the collagen delivery and moisture aids and surface protection from the environment.

As may be appreciated, the above aspects produce a new class of multi-layered wound dressing mesh based on electrospun nanofibre to provide both absorption or moisture for macro-environment protection and with chemotactic properties to promote angiogenesis and cell remodelling. The mesh layer may be manufactured out of single use non-dissolvable biodegradable polymers such as, but not limited to electrospun non-dissolvable collagen, Poly caprolactone (PCL), Poly lactic glycolic acid (PLGA), Poly Lactic acid (PLA), Poly vinyl butyral (PVB), gelatin or chitosan nanofibres or their combinations, layered with dissolvable denatured marine collagen nanofibres to advantageously dissolve in wound fluid instantaneously and release therapeutic actives to promote haemostasis, angiogenesis and remodelling. Furthermore, the non-dissolvable nanofibres will remain outside the wound bed to promote haemostasis by providing moisture. Advantageously, another layer of slow dissolving mesh can be incorporated in the matrix with therapeutic bioactives to support cell proliferation during the second phase of wound healing. The method of manufacture allows for the non-dissolvable layer of the nanofibre to be co-spun with the dissolvable polymer or be a composite layer with a dissolvable polymer. Likewise, the slow dissolving and the fast dissolving polymer can be co-spun together as a separate composite layer. Advantageously, all these layers can carry multiple actives to release at different rates to target multiple cells of the skin. Furthermore, the composition comprises predominately 'Type I' and, advantageously is denatured whole chain collagen (DWC), not gelatin. Therefore, the composition has a structure closer to the natural triple helix collagen structure found in human skin. Finally, the Applicant's composition fulfils a market need for a marine-sourced 'Type I' and denatured collagen minimizing the possibilities of transmitted diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

Further aspects of the multilayer matrix nanofibre composition, bioactive compositions and their usage will become apparent from the following description that is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 illustrates a schematic representation of the complete wound healing cycle as is known in the art;

Figure 2 illustrates a schematic representation of the working mechanism of the present invention on different layers of the skin;

1) Top or third layer: a cover substrate (optional)

2) First layer: Bio-degradable nanofibre impregnated with a high loading of actives

3) Second layer: Rapid dissolving collagen nanofibre carrying actives

4) Keratinocyte

5) Epidermal stem cell

6) Melanocyte

7) Dermal stem cell

8) Fibroblast cell

9) Endothelial cells Figure 3 illustrates the A) graphical representation of present invention on wounded skin, B) SEM image of the top and first layer (PCL), and C) SEM image of the second layer (type I denatured marine collagen);

10) Top or third layer: a cover substrate (optional)

11) First layer: Bio-degradable nanofibre impregnated with a high loading of actives

12) Second layer: Rapid dissolving collagen nanofibre carrying actives

13) Epidermis

14) Dermis

15) Flair follicle

16) Hair

17) Sweat duct

18) Sebaceous gland

19) Sweat gland

20) Fat

Figure 4 illustrates the type I molecular profile of marine collagen nanofibres on SDS page gel;

Figure 5 illustrates Franz cell-diffusion study performed on human skin biopsy of different thickness A)

Epidermal representation - Thin skin (~ 500 urn), B) Epidermal -dermal junction representation - Middle skin (1.22 mm) and C) Dermal representation Thick skin (1.8 mm);

Figure 6 illustrates Optical Coherence Tomography (OCT) data showing the Marine collagen nanofibre (MCN) penetration into the skin. A) at 0 min, the MCN penetrated to 1.5 mm depth of the skin and moves to 1.75 mm in 18 min. B) at 35 min the MCN penetrates deeper to reach 2 mm, C) at 50 min MCN moves deeper towards 2.5 mm, D) combined OCT data of A, B, and C showing the total penetration profile of OCT over a period of time;

Figure 7 illustrates the real-time OCT image data showing MCN penetration into pitted skin at A) 0 min and B) 50 min; and

Figure 8 illustrates Optical Coherence Tomography (OCT) data showing the penetration of bio actives carried by first layer (example PCL) into the skin.

DETAILED DESCRIPTION

As noted above, described herein is a multilayer nanofibre polymer composition comprising non-woven mesh including one or more bioactive compounds. In particular, the mesh comprises one or more polymer nanofibres with different properties. The therapeutic bioactives may be chemically or physically bonded with the nanofibre matrix. Upon exposure to wet or moist skin, one or more of the nanofibres may dissolve rapidly thereby releasing actives, while the other layer (s) of nanofibre polymers may remain and/or degrade over a further period of time, thereby releasing further actives. For the purposes of this specification, the term 'about' or 'approximately' and grammatical variations thereof mean a quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% to a reference quantity, level, degree, value, number, frequency, percentage, dimension, size, amount, weight or length.

The term 'substantially' or grammatical variations thereof refers to at least about 50%, for example 75%, 85%, 95% or 98%.

The term 'comprise¹ and grammatical variations thereof shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements.

The term 'one or more active agents' or 'one or more bioactives' and grammatical variations thereof include analogues of active agents.

The terms 'bioactive' and 'agent' or grammatical variations may be used interchangeably for the purposes of this specification referring to a compound with an activity in vivo or in vitro that may be cosmetic or medical. Where the singular or plural of each word is used, the opposite may apply and use of the singular or plural for either term should not be seen as limiting.

The term 'analogues of active agents' or grammatical variations thereof refers to an active agent having a structure, function, and/or composition equivalent to that of the agent in a native or natural un extracted form. Exemplary methods to determine functional equivalence of active agents, such as those obtained from plants and their analogues are well known in the art and representative methods are provided herein in the Examples.

As used herein the term 'dressing' or grammatical variations thereof includes patches, strips, bandages or plasters.

As used herein the term 'promoting' or grammatical variations thereof includes initiating, enhancing or mediating, for example promoting a biological response includes initiating, enhancing or mediating a biological response.

The term 'effective amount' or grammatical variations thereof with reference to an amount or dosage of the composition described herein refers to an amount of a composition which is sufficient to effectively cause the described action such as preventing, treating or reducing a condition, the condition being a cosmetic change or medicinal change.

The term 'stable' or grammatical variations thereof refers to the active agent or agents not undergoing substantial physical changes, chemical changes, microbial growth, or any substantial loss in activity over time when stored in the absence of moisture.

The term 'natural' or 'natural based' and grammatical variations thereof refers to compounds obtained from nature or one or more synthesised versions of compounds found in nature. Ideally, the compound or compounds used meet the Natural Products Association (NPA) guidelines i.e. they are derived from renewable sources in nature' they do not use petroleum compounds, they meet generally recognised as safe or GRAS standard as set by the USA FDA and they are manufactured based on NPA approved processes.

The term 'dry' and grammatical variations thereof refer to a water activity sufficient to impair or stop microbial activity.

The term 'penetration' and grammatical variations thereof refer to actives and/or nanofibre dissolving and migrating into the skin layers.

The term 'absorbtion' and grammatical variations thereof refer to actives and/or nanofibre absorbing into the skin layers.

The term 'moisture' and grammatical variations thereof refers to the presence of a hydrating fluid such as water in sufficient amounts to at least support microbial growth.

The term 'dissolve' and grammatical variations thereof with reference to a solid such as a nanofibre, refers to the solid becoming a liquid or being incorporated into a liquid so as to form a solution.

In a first aspect there is provided a multilayer matrix nanofibre composition for wound healing management comprising: at least two nanofibre layers; and an effective amount of at least one bioactive compound physically and/or chemically bonded within the nanofibre layer matrix; the bioactives in at least one layer are segregated from bioactives in at least one other layer preventing any reactions between the bioactives in each layer during storage of the composition under ambient conditions substantially free of moisture; wherein the first layer nanofibre is formed from a base material that is solubilised with the bioactives and electrospun together or the bioactives are coated on the nanofibre surface via spray coating or dip coating; wherein the second layer nanofibre is formed from a base material that is solubilised with the bioactive or bioactives in an aqueous-based solvent solution and the base material and bioactives are together spun via electrospinning to form dry fibres; and wherein on exposure to moisture, at least one of the nanofibre layer dissolves and/or degrades to slowly to release actives over a period of time and/or to act as a sacrificial protective skin layer, and the other nanofibre layer(s) dissolves rapidly, thereby substantially releasing all of the at least one bioactive compound.

The composition may comprise one or more polymer nanofibres with therapeutic bioactives for wound healing, wherein the one or more nanofibres may degrade slowly and act as sacrificial protective skin layer and other nanofibre may dissolve on a wet skin immediately thereby releasing actives.

The dissolvable polymer and the carried therapeutic actives may assist in haemostasis and collagen remodelling. In this way, the multilayer polymer compositions with different functional actives may function as a wound healing agent when administrated to the target area. Such compositions have been found to be particularly well suited for use in a variety of wound management scenarios. For example, acne wound and atrophic scars, post-treatment wound care for surgical and burn wounds, diabetic wound dressings or where immediate medical intervention is highly critical.

The top layer of the compositions of the present invention may be configured as a protective layer over the existing wound. In this way, the top layer and the first layer is strong, stretchy, porous and hydrophilic.

The first layer of the compositions of the present invention may also be configured as a protective layer over the existing wound in addition to promoting proliferation of cells by releasing actives sustainably .

In this way, the layer is strong, having pores with high surface area to absorb any exudate and release MMPs regulators, while the functional actives/agents embedded in the first layer may promote the other phases of the wound healing process.

The second layer of the compositions may be manufactured from collagens with a different molecular weight, length and different penetration efficiency. For example, the second layer may be a mix of denatured whole chain and collagen peptides with hydroxyl groups. These two different kinds of collagens are essential to carry different actives or nutrients to perform different activities at the same or different layers of the skin at the same time.

The second layer of the present invention may dissolve immediately on the wound to release actives to promote haemostasis to coagulate over bleeding and the re-epithelialization at the inflammation stage. The working model of this layer is tested using penetration and absorption of electrospun caffeine drug in a collagen matrix on a skin biopsy with different thickness as shown in Figure 5A -C.

In other embodiments, other layers of the composition may be configured to work synergistically together to manage the full wound healing cycle. In fact, the inventors have discovered a new generation of the wound dressing for faster wound healing process when multi-layered marine collagen nanofibre with therapeutic actives are administrated to injuries. The invention is based on the difference within the penetration efficiency of each layer in the created multi-layered collagen nanofibre mesh.

The penetration efficiency also may be determined by Optical Coherence Tomography (OCT) techniques, known by those skilled in the art and as shown in Figures 6, 7 and 8.

Previous studies have shown the ability of collagen derived fragments to control many cellular functions, including cell shape and differentiation, migration, and synthesis of many proteins. As aforementioned, collagen-based prior art wound dressings are either designed for wound exudate management or to aid granulation tissue formation and epithelialization. Therefore, two dressings i.e. primary and secondary dressings have to be utilised to treat a patient's wound cycle. Moreover, the type of wound acute or chronic can increase the complexity and changing intervals of primary and secondary dressings. Advantageously, the present invention requires only one multi-layered nanofibre dressing that may be comprised of marine collagen. In this way, the Applicant's dressings may fulfil all the attributes such as hydrophilicity, high surface area, customized pore size, high breathability to cover all the four phases of wound healing and to meet specific treatment goals including support of granulation tissue formation, management of bioburden and support of wound closure.

For an active wound recovery, it is necessary to modulate matrix metalloproteases (MMPs). In chronic wounds, the MMPs are higher than those of acute injuries. Therefore, chronic wounds and acute injuries require different MMP regulation regimes for these different types of injuries. Currently, there are two methods in which wound dressing material aims to modulate MMP activity. The first method is through the absorption of wound exudate, simultaneously removing MMPs from the wound bed into the dressing. The second method is by binding proteases to the dressing or directly inactivating the MMPs. The most identified MMPs for wound healing was MMP-1, MMP-2, MMP-8, and MMP-9. Besides, some wound dressing product has an antimicrobial component added.

The Applicant's consider that the novel multi-layered nanofibre composition may provide multiple benefits and has dual functionality to heal wounds. In this way, the biodegradable non-dissolvable nanofibre may provide moisture control and absorb wound exudate during the haemostasis and inflammation phase, and the nanofibres may act as a sacrificial substrate and provide an alternate source of collagen that can be degraded by high levels of MMPs; Thus, accelerating the process granulation tissue formation during the proliferation phase. Without being bound by theory, the fast dissolving second layer may immediately release actives to initiate blood coagulation and provide nutrients for the wound bed. Furthermore, regulating the MMPs levels may allow the endogenous native collagen in the wound bed to continue with the normal wound healing pathway. In this way, the present invention may provide control over cellular functions at each phase of wound healing.

It is well known that cell contact with Extracellular matrix (ECM) molecules influence cell behaviour by dictating the quality and quantity of matrix deposition. Type I collagen is the most abundant structural component of the dermal ECM matrix. ECM has differing effects on keratinocyte motility. Signalling through receptors and secretion of collagenase are both components of this process. With continuing research, understanding the working of ECM on keratinocyte migration may assist in the development of therapeutic effects to support re-epithelialization to enhance wound healing artificially.

Keratinocytes are known to recognize and migrate on Type I collagen substratum, resulting in collagenase production. Collagenase, in turn, promotes efficient migration of keratinocytes over the dermal and provisional matrix. Research has shown that although keratinocytes adhere to gelatin (denaturated collagen), collagenase production is not turned on in response to its substrate, which supports the preference for wound dressings to be wholly or partially manufactured of Type I collagen. In preferred embodiments, collagen sourced from marine may be disentangled a-chains of high molecular weight (HMW) denatured whole chain collagen (DWCC) with intact telopeptides. Advantageously this unique formation has been found to be closer to the native triple helix structure of Type I and is structurally different to gelatin. Advantageously, the molecular format of bulk collagen does not change after electrospinning and remains in nanofibre form. In this way, the intact telopeptides present in DWCC nanofibre may provide active sites to carry actives or drugs specific to wound healing while the hydrophilic nature of the marine collagen may work on cellular adhesion, simultaneously encouraging mobility of granulocytes, macrophages, and fibroblasts. Furthermore, the collagen nanofibre may promote the migration of keratinocytes to migrate from the edge of the wound across the whole area of tissue during proliferation to achieve support effective re-epithelialization. Moreover, the DWCC itself may expose a higher proportion of the polypeptide sequences to potential interactions with wound site components, thereby improving healing capacity.

As noted above, the nanofibres described herein dissolve in the presence of moisture. In one embodiment, the nanofibres dissolve and release substantially all of the bioactives within about 0-50 minutes when moistened. The dissolution rate may be a rate of at least 2-100 %/min when solubilised in water at a temperature of about 5-30 °C.

The therapeutic actives bound to the non-dissolvable electrospun polymer may immediately release or slowly release over a period of time.

In other embodiments, the therapeutic actives bound to the non-dissolvable electrospun polymer configured to release within several weeks.

Dissolution, particularly at variable simultaneous rates for a multilayer matrix noted represents a significant change to the art where for example set rates of dissolution occur with only one layer. In the art, bioactives if present are typically only released from one fibre matrix at a time.

The nanofibres may be tailored to release substantially all of one or more of the active agents to which it is associated or bound within about 0-3000 minutes, 1-4 weeks, and/or 1-12 months, when applied to skin.

The temperature of solubility may range from 15, or 16, or 17, or 18, or 19, or 20, or 21, or 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30°C. As noted above, the speed of dissolution may occur on skin and at skin temperatures of around 15-30°C. Topical delivery is a preferred application hence the composition must be designed to maximise delivery or dissolution at skin temperatures. Typically, higher temperatures may promote faster dissolution however, in the present case, temperatures above 30°C are undesirable due to the unpleasant effect of higher temperatures on the skin as well as the need to then heat the composition prior to use.

As noted in the above embodiment, at least one layer is made from a nanofibre material that dissolves at a different rate to a second or further layer, thereby releasing the bioactive(s) at different rates. As may be appreciated, segregating bioactives in a single delivery vehicle may be advantageous as it avoids the bioactives reacting together during storage yet allows co-administration, avoiding the need to apply two different products. At least from a convenience point of view, providing one product that delivers multiple bioactives would be of benefit particularly if the bioactives were normally incompatible.

The first nanofibre and the at least one further nanofibre may be the same material. Alternatively, the first nanofibre may differ to at least one or more of the nanofibres used in the further layer or layers. By way of example, a multilayered matrix may be formed from a first layer made from polycaprolactone (PCL), or PCL co-spun with type I collagen nanofibres bonded with a first bioactive, and a second layer of the matrix, formed from type I collagen nanofibre, with a different bioactive bonded thereon.

The dissolvable polymers may have different types of long chain and short chain polymers, wherein the long and short chain polymers may be co-spun to form an interpenetrated network or chemically linked together.

Preferably, both short and long chain dissolvable electrospun polymers may have a cross-sectional diameter of approximately lum to 100 nm.

Preferably, both short chain and long chain dissolvable natural polymers may be manufactured from a polymer solution selected from any one of: collagen, whole chain collagen, denatured collagen, denatured whole chain collagen, collagen peptides, gelatin, polyvinyl alcohol (PVOH or PVA), polyethylene oxide (PEO), alginate, marine algaes in general, ucoidan, Chitosan, gelatine, dextran, pullulan, polysaccharides generally, and/or combinations thereof.

The composition may comprise nanofibres formed from collagen protein.

In one embodiment, the second electrospun polymer (e.g. first layer) may be configured to stay on the skin or degrade within several weeks.

Preferably, the non-dissolvable polymers may be manufactured from a polymer solution selected from any one of polycaprolactone (PCL), crosslinked polyvinyl alcohol (PVOH or PVA), polyamide 6 (PA-6), polyvinyl butyral (PVB), polyamide 6,6 (PA-6,6), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), alginate, collagen, collagen peptides, gelatin, Fucoidan, Chitosan, gelatine, dextran, pullulan, marine algaes, polysaccharides generally, carboxymethyl cellulose (CMC), polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and/or combinations thereof.

The nanofibres in each layer may form a homogenous or heterogeneous matrix of nanofibres. A homogenous matrix of nanofibres is one in which the matrix layer or layers may be formed from a single nanofibre. A heterogeneous matrix of nanofibres is one in which the matrix is formed from multiple different nanofibres.

Nanofibres formed from one polymer or protein may be associated together, for example using van der waals forces, to form a layer, and nanofibres formed from a different polymer or protein may be associated together to form another layer. Each layer may be associated with the other, for example by van der waals forces. For example, in one case of a multilayer nanofibre matrix formed from collagen and PCL or PVOH, the collagen nanofibres form a layer where the collagen nanofibres are associated with each other, for example by van der waals forces, and the PCL or PVOH nanofibres form an associated layer.

In one embodiment, the multiple dissolvable polymers and non-dissolvable with multiple actives may be layered to form composites.

More preferably, the two electrospun polymers with multiple actives may be electrospun together to form a single layer of nanofibre co-polymer.

Preferably, the therapeutic actives may be bonded with at least one of the polymer nanofibres. In this way, the therapeutic actives bonded with non-dissolvable polymer nanofibre may function during the inflammatory, angiogenesis, and haemostasis phases.

Preferably, the non-dissolvable electrospun polymer and the top layer mesh may be configured to function as a physical barrier to prevent infection.

In alternative embodiments, the non-dissolvable electrospun polymer may or may not contain therapeutic actives.

In one embodiment, the non-dissolvable polymer may contain actives such thrombin, fibrinogen, ceramids, antibodies, antigens, peptides, alginates, collagen, gelatin, polyphenols like catechins and flavonoids, carotenoids, proteins, caffeine, ketones, fatty acids, glycerides, ceramides, carbohydrates, enzymes, serine proteases generally, Cu+, Mg+, Ca+, metal ions in general, metal-organic frameworks (MOFs), phospholipids, curcumin, terpenes, manuka tri-ketones and/or combinations thereof.

In one embodiment, the dissolvable polymer may contain actives such alginates, phyto-compounds, polyphenols, vascular endothelial growth factors (VEGFs), EDTA, metal chelators, Glycosaminoglycans (GAG's) like hyaluronic acid, curcumin, terpenes, manuka tri-ketones generally, collagen, peptides, terpenes, gelatin, polyphenols like catechins and flavonoids, carotenoids, proteins, caffeine, ketones, fatty acids, glycerides, ceramides, carbohydrates, enzymes, serine proteases generally, cationic nanocylinders, Cu+, Mg+, Ca+, metal ions in general, metal-organic frameworks (MOFs), phospholipids, squalene, monoglycerids and triglycerids and/or combinations thereof.

The therapeutic actives bound to the non-dissolvable electrospun polymer may function as a sacrificial layer, but release actives.

Preferably, the non-dissolvable electrospun polymer may have a cross-sectional diameter of approximately lum to 100 nm.

As noted above, the nanofibre material may be varied to dictate the rate of release of the active. Polymers and proteins may dissolve at varying rates. In one embodiment, the first layer may be made from a nanofibre that releases a bioactive at a first speed while a second layer may release a second bioactive at a second speed. The second speed may be slower than the first speed. The second speed may be faster than the first speed. One example of this application may be in delivery of a bioactive compound that requires activation or catalysation via an enzyme. The first layer dissolves rapidly to release inactive bioactive on to skin and then, at a slower rate, the second layer of nanofibre dissolves releasing an active enzyme that catalyses conversion of the inactive bioactive to an active state. As may be appreciated, a multilayer composition as described may be helpful as it ensures conversion of the inactive bioactive and does this at the actual site where the bioactive is needed (for example, the skin). This means minimal loss in activity hence a known and repeatable dosage.

It should be appreciated that the targeted selection of bioactive and nanofibre enables the production of a multilayer nanofibre matrix having multiple kinetics of bioactive release, for example wherein one or more bioactives may be rapidly released from one nanofibre present in the matrix, such as to give an antibacterial or antimicrobial effect, and one or more bioactives may be released over a longer period, for example to support skin repair, recruit immune cells to the site of application, or the like.

The embodiments described above may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more said parts, elements or features.

Further, where specific integers are mentioned herein which have known equivalents in the art to which the embodiments relate, such known equivalents are deemed to be incorporated herein as if individually set forth.

WORKING EXAMPLES

The above-described multilayer matrix nanofibre composition, bioactive compositions, and their usage are now described by reference to specific examples.

EXAM PLE 1

The multilayer of the matrix nanofibre composition is designed to work synergistically together to manage the full wound healing cycle as shown in Figure 1.

The wound healing cycle/process can be divided into four overlapping phases a) haemostasis; b) inflammation; c) proliferation; and d) wound remodelling.

Haemostasis: Haemostasis is an express process where duration is generally measured in minutes and occurs at the wound site. The pore size of the dressing is essential to allow cells to enter the dressing and concentrate therein. Additionally, surface area plays a significant role in exudate. The larger the surface area, then more the exudate is absorbed.

It has been shown that the slow-degrading first layer nanofibre of the present invention with a high surface area relative to low pore size assists to protect the wound area from environmental exposure and exudate wound. While actives such as serine protease, growth factors (GFs) or any anti-microbial agents stimulate a variety of inflammatory cells carried by the faster-dissolving second layer, they also act within minutes to stimulate the intrinsic clotting cascade, initiate the inflammatory phase and provide a moist environment. The slow degrading first layer of the present invention can be made of PCL or non-dissolvable collagen or both. The OCT results of an example of the first layer made of PCL (Figure 8) prove the delivery of actives into the skin.

Inflammation: The inflammation process generally takes days for completion. Naturally, the action of proteolytic enzymes secreted by inflammatory cells on the collagen present in the ECM gives rise to many peptides during wound healing. These peptides have a chemotactic effect on the recruitment of other inflammatory cells such as neutrophils, macrophages, etc. Activated macrophages produce pro- inflammatory cytokines such asTNF-a and IL-Ib, which directly influence the deposition of the collagen in the wound by inducing synthesis of collagen via fibroblasts and regulation of tissue inhibitors of matrix metalloproteinase (TIMPs).

Studies have shown that a wet or moist environment in wounds reduced the inflammatory reaction achieving the fastest healing with fewest aberrations and scar formation. The Applicant has found that the slow degrading first layer of the present invention possesses hydrophilic properties having a high surface area that retains moisture during the healing process. This creates a wound environment favourable for cell multiplication. As the cellularity of the wound increases, the proliferative phase begins.

Proliferation: The cleavage products resulting from collagen degradation stimulate fibroblast proliferation, and it has shown to take weeks for the process to be completed. Collagen plays a vital role in this phase. As aforementioned, high secretion of MMPs needs to be regulated to promote the formation of ECM. MMPs degrade non-viable collagen in order to prepare the wound dressing. The degraded collagen works as chemotactic agents to signal fibroblast and endothelial cells to proliferate.

The slow-degradable collagen in the present invention provides a moist environment but also serves as a sacrificial layer for the degradation by MMPs when placed on the wound. The nanofibre structure matrix mimics the natural ECM structure to effectuate its action as a sacrificial layer. This artificial degradation signals the other cells, such as fibroblasts and endothelial cells for the formation of the granulation tissue. As the first layer of collagen dressing is degraded, the MMPs are released back into the wound. These MMPs are de-activated by the actives encapsulated in the first layer of collagen dressing, while the peptides or drugs released by the second layer work on re-building collagen and provide nutrients to favour re-modelling. Thus, it has been found the novel multilayer composition accelerates the proliferation process and restores the balance of MMPs while protecting the wound from environmental vulnerability. The obtained OCT (Figure 7) and Franz cell results (Figure 5A, B and C) provides proof of the rapid and sustained drug release function of the second layer. In addition, the collagen matrix of first and second layer proves to have no cytotoxic effects on human keratinocytes and fibroblast cells (Table 1).

Table 1 illustrates the cytotoxicity results of marine collagen nanofibre of the present invention on human keratinocytes and fibroblast cells. The results prove that there is no evidence of a cytotoxic effect of the present invention on those cells over 72 hours. These results were determined using the Lactate dehydrogenase (LDH) method.

Remodelling: Once the balance between the production of scar matrix and their degradation by MMPs is regulated, the remodelling i.e. the creation of new skin begins. It has been found that the duration of the remodelling process can be upwards of 1+ years. The remodelling phase requires a macro environment suitable for the maturation of scars and regular supply of essential nutrients. It has been found that the present invention fulfils these requirements as it carries essential drugs or nutrients to supply the scar matrix and breathable pore layer promotes faster skin recovery when applied regularly.

Remodelling of atrophic scars: Atrophic acne scars is a permanent complication of acne vulgaris and common acne scars conditions of the adolescent population. The pathogenesis of atrophic scarring is related to inflammatory mediators and enzyme degradation of collagen type I. Therefore, most of the common re-modelling or acne scar clearing treatment focuses on improving collagen production. In fact, the use of collagen fillers for atrophic acne scars is recommended by the American Society for Dermatologic Surgery. Given the fact, the current invention carries the collagen matrix as the primary vehicle for drug delivery and providing moistening for faster healing; it can be a great alternative and effective treatment for atrophic acne scarring.

EXAMPLE 2

With reference to Figure 3A, a graphical representation of the construction of the present invention on wounded skin is shown. Figure 3B shows an SEM image of the top and first layer (PCL), and Figure 3C an SEM image of the second layer (type I denatured collagen).

The SDS page gel of the type I collagen nanofibre of the second layer of the present invention is shown in Figure 4. In particular, Figure 4 shows the SDS page analysis of pure marine collagen raw material and lines 3 and 4 (two different batches) and marine collagen nanofibres line 2. The molecular weight distribution in lines 3 and 4 is typical to type I collagen. Line 2, the nanofibres with similar weight distribution proves the genetic variation is unaffected by electrospinning and the nanofibres remain as type I in nanofibre format.

EXAMPLE 3

Franz-cells diffusion is a widely accepted methodology to evaluate in-vitro drug permeation. Here the selected model drug caffeine penetration was performed on a human skin biopsy with three different formulations: A) caffeine in solution form, B) caffeine in gel form, and C) caffeine electrospun with a collagen nanofibre patch. The caffeine solution was obtained from Sigma, 5% caffeine percutafeine was used as a gel, and caffeine nanofibre was electrospun at the laboratory. Phosphate buffer saline (PBS) was used as the analyte solvent.

Three different skin thicknesses were selected for this study. One excised abdominal skin (operative waste) was obtained from a surgery procedure and was used as soon as possible after a patient's excision. The excised abdominal skin was cut into nine fragments and was placed immediately in a Franz cell system. The abdominoplasty skin was obtained from three women aged 35, 40 and 45 respectively.

Figures 5A, B and C respectively show the penetration of caffeine on three different human skin layers over a period of one day. The efficiency was compared to a solution form of caffeine and a gel from caffeine. The skin fragments of different thickness were placed on the system where the skin thickness was designated as:

• Thin skin (around 500 pm),

• Middle skin (1.2 mm),

• Thick skin (1.8 mm).

Once the caffeine formulation was added on to the test samples, the samples were collected after 15 min, 30 min, 45 min, lh, lh 15 min, lh 30 min, 4h, 8h, and 24h after the deposit of caffeine. The samples were frozen at minus 80°C until analysis.

After 1 hour of stabilization following the cutting of the skin and the system assembled, the Franz cell experiment was started.

Thickness of the skin: 517 mhΊ

The thickness of the skins was 584 pm, 461 pm and 506 pm for the solution, the gel, and the patch respectively. In Figure 5A, there was no difference in the amount of caffeine remaining on the skin surface. Still, the caffeine penetration was increased after 24h with the nanofibre compared to the gel and solution. This complements the OCT results shown in Figures 3 and 4, thus proving the nanofibre patch is more suitable for sustainable drug release, further demonstrating that the second layers of the present invention can carry drugs or actives for sustainable release over a period. In practice, this will minimize the routine for changing the dressing patch.

Thickness of the skin: 1.22 mm

The thickness of the skins was 1.17 mm, 1.28 mm, and 1.22 mm for the solution, the gel and the patch respectively. The caffeine penetration was increased with the patch compared to gel and solution even at 0 min. Again, this complements the OCT results showing immediate penetration of actives into the skin (see for example, Figure 5B). Furthermore, there was a continuous increase in the detection of caffeine in that layer, which is the dermal-epidermal junction (DEJ) or dermal layer in some part of the human body. DEJ consists of interconnected proteins in a complex network which helps for remodelling, healing of wounds, and development of the new skin. Hence, the present invention proves the penetration of drugs at this thickness of the skin is fast, continuous, and sustainable. Therefore, the second layer of the present invention with appropriate drugs for remodelling would conceivably work for remodelling of the skin and specifically for acne scarring.

Thickness of the skin: 1.78 mm

The thickness of the skins was 1.73 mm, 1.78 mm, and 1.84 mm for the solution, the gel and the patch respectively. In Figure 5C, the caffeine penetration was increased at 8h and 24h with the patch compared to gel and solution. Depending on the part of the human body, 1.5 to 4 mm generally consists of dermal components. The dermal part contains three major proteins situated in an extra fibrillar matrix: collagen, mucopolysaccharides, and elastin. The present invention shows a continuous increase in the detection of caffeine in that dermal layer. Therefore, the building of ECM, regulating MMPs require sacrificial collagen and actives that can stimulate collagen production. Thus, the first layer of the present invention when made of a non-dissolvable Type I collagen matrix can support the production of collagen at the dermal layer.

EXAMPLE 4

The penetration efficiency of the multilayer matrix nanofibre composition wound dressing and associated bioactives and working mechanism as described above is based on OCT and Franz-cell diffusion testing results.

The second fast-dissolving layer carrying drugs as shown in Figure 7 was subjected to the OCT test.

The OCT has been proven to be the optimal scientific method/technique for a non-invasive and non destructive functional real-time spectral imaging technique that can provide 2D and 3D images in vitro and in vivo. The methodology, experimental design, and testing protocols are strategically intended to visualize the bioactives delivery, and penetration into an exemplary piglet skin tissue.

With reference to Figure 7, the degree of penetration of the bioactive in each nanofibrous matrix is illustrated. Although the human skin morphology and layer thickness varies from person-to-person, it is widely understood by those skilled in the art, the average thickness of an epidermis layer is 0.6 pm, and the dermal layer may vary between 2 - 4 mm from the top stratum corneum.

It was established through the OCT profile that the collagen nanofibre penetrates deeply, crossing the stratum corneum and reaches a depth of approximately 1.5 mm up to the dermal layer of the skin instantly, i.e., at 0 min, and moves further down the skin layer (1.75 mm) in 18 min. Therefore, the fast dissolving second-layer of the present invention could carry actives like thrombin to promote blood coagulation.

It is documented that the size of the molecule and its lipophilicity are the major determinants of the penetration processes through the stratum corneum. This means that the permeability of a molecule is directly related to its hydrophilicity or lipophilicity and inversely proportional to its molecular size. Most drug formulations use skin penetration enhancers or encapsulations to help promote the transport of actives into the skin, with water being the safest and most effective skin penetration enhancer with high permeability constant 5.5 x 10⁶ cm/hr. Therefore, simple hydration of the stratum corneum can modify the surface efficiently to promote penetration of the actives.

The second layer of the present invention, the DWCC nanofibre matrix being a water-soluble material naturally diffuses through the stratum corneum pores on a wet surface (due to its hydrophilicity) and promotes the transport of actives attached to it, which is best seen in Figure 6A.

Over the next 35 minutes, the nanofibres cross 2 mm of the skin. The actives continue to penetrate further and reach 2.5 mm in 50 minutes which is a dermal layer of the skin (Figure 6C). This layer consists of fibroblast connective tissues to produce natural collagen and other fibres. Therefore, the results indicate that the present invention could conceivably be used to reconstruct ECM on the skin with suitable actives.

Figure 6D is the overlap of figure 6A to C, showing the penetration fashion of actives into the skin.

The penetration fashion of bioactives was evidently different to the same bioactives carried in the dissolvable nanofibre. From 0 min to 35 min there was very little difference in the penetration depth proving the slow kinetics of bio-actives carried through non-dissolvable nanofibre matrix. Therefore, the slow dissolving first layer of the present invention can carry MMP regulators and the nanofibre matrix can itself become a scaffold for granulation.

As above, Figure 7 shows the real -time OCT image of skin of the second layer. The skin with a pit was selected to represent an atrophic acne scar. The intense red colour representing the presence of collagen matrix on the surface at 0 min moves deeper inside through the pit and becomes absorbed by the skin. Thus, proves the present invention is suitable for treating atrophic acne scar conditions. The first non-dissolvable constructed using PCL with bioactives were also subjected to OCT analysis.

Aspects of the present invention have been described by way of example only, and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the description and claims herein.

Claims

WHAT IS CLAIMED IS:

1. A multilayer matrix nanofibre composition for wound healing management comprising: at least two nanofibre layers; and an effective amount of at least one bioactive compound physically and/or chemically bonded within the nanofibre layer matrix; the bioactives in at least one layer are segregated from bioactives in at least one other layer preventing any reactions between the bioactives in each layer during storage of the composition under ambient conditions substantially free of moisture; wherein the first layer nanofibre is formed from a base material that is solubilised with the bioactives and electrospun together or the bioactives are coated on the nanofibre surface via spray coating or dip coating; wherein the second layer nanofibre is formed from a base material that is solubilised with the bioactive or bioactives in an aqueous-based solvent solution and the base material and bioactives are together spun via electrospinning to form dry fibres; and wherein on exposure to moisture, at least one of the nanofibre layer dissolves and/or degrades to slowly to release actives over a period of time and/or to act as a sacrificial protective skin layer, and the other nanofibre layer(s) dissolves rapidly, thereby substantially releasing all of the at least one bioactive compound.

2. The composition as claimed in claim 1, wherein the composition comprises one or more polymer nanofibres with therapeutic bioactives for wound healing, wherein the one or more nanofibres may degrade slowly and act as sacrificial protective skin layer and other nanofibre may dissolve on a wet skin immediately thereby releasing actives.

3. The composition as claimed in claim 2, wherein the polymers and carried therapeutic actives assist in haemostasis and collagen remodelling.

4. The composition as claimed in any one of the preceding claims, wherein the first or top layer of the composition is configured as a protective layer over an existing wound in addition to promoting proliferation of cells by releasing actives sustainably.

5. The composition as claimed in any one of the preceding claims, wherein the second layer of the composition is manufactured from collagens with a different molecular weight, length and different penetration efficiency.

6. The composition as claimed in claim 5, wherein the second layer dissolves immediately on the wound to release actives to promote haemostasis to coagulate over bleeding and the re- epithelialization at an inflammation stage.

7. The composition as claimed in any one of the preceding claims, wherein collagen sourced from marine is disentangled a-chains of high molecular weight (HMW) denatured whole chain collagen (DWCC) with intact telopeptides.

8. The composition as claimed in any one of the preceding claims, wherein the nanofibres dissolve and release substantially all of the bioactives within about 0-50 minutes when moistened.

9. The composition as claimed in any one of the preceding claims, wherein dissolution rate is a rate of at least 2-100 %/min when solubilised in water at a temperature of about 5-30 °C.

10. The composition as claimed in any one of the preceding claims, wherein the therapeutic actives bound to the non-dissolvable electrospun polymer immediately release or slowly release over a period of time.

11. The composition as claimed in claim 10, wherein the therapeutic actives bound to the non- dissolvable electrospun polymer are configured to release within several weeks.

12. The composition as claimed in claims 1 to 9, wherein the nanofibres release substantially all of one or more of the active agents to which it is associated or bound within about 0-3000 minutes, when applied to skin.

13. The composition as claimed in claim 11, wherein the nanofibres release substantially all of one or more of the active agents to which it is associated or bound within about 1-4 weeks when applied to skin.

14. The composition as claimed in claims 1 to 9, wherein the nanofibres release substantially all of one or more of the active agents to which it is associated or bound within about 1-12 months, when applied to skin.

15. The composition as claimed in any one of the preceding claims, wherein at least one layer is made from a nanofibre material that dissolves at a different rate to a second or further layer, thereby releasing the bioactive(s) at different rates.

16. The composition as claimed in claim 15, wherein a multilayered matrix is formed from a first layer made from polycaprolactone (PCL), or PCL co-spun with type I collagen nanofibres bonded with a first bioactive, and a second layer of the matrix, formed from type I collagen nanofibre, with a different bioactive bonded thereon.

17. The composition as claimed in any one of the preceding claims, wherein the dissolvable polymers have different types of long chain and short chain polymers, wherein the long and short chain polymers are co-spun to form an interpenetrated network or chemically linked together.

18. The composition as claimed in claim 17, wherein both short and long chain dissolvable electrospun polymers have a cross-sectional diameter of approximately lum to 100 nm.

19. The composition as claimed in claim 17 or claim 18, wherein both short chain and long chain dissolvable natural polymers are manufactured from a polymer solution selected from any one of: collagen, whole chain collagen, denatured collagen, denatured whole chain collagen, collagen peptides, gelatin, polyvinyl alcohol (PVOH or PVA), polyethylene oxide (PEO), alginate, marine algaes in general, ucoidan, Chitosan, gelatine, dextran, pullulan, polysaccharides generally, and/or combinations thereof.

20. The composition as claimed in any one of the preceding claims, wherein the nanofibres are formed from collagen protein.

21. The composition as claimed in any one of the preceding claims, wherein non-dissolvable polymers are manufactured from a polymer solution selected from any one of: polycaprolactone (PCL), crosslinked polyvinyl alcohol (PVOH or PVA), polyamide 6 (PA-6), polyvinyl butyral (PVB), polyamide 6,6 (PA-6,6), polyethylene terephthalate (PET), polybutylene terephthalate (PBT), polylactic acid (PLA), poly(lactic-co-glycolic) acid (PLGA), alginate, collagen, collagen peptides, gelatin, Fucoidan, Chitosan, gelatine, dextran, pullulan, marine algaes, polysaccharides generally, carboxymethyl cellulose (CMC), polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and/or combinations thereof.

22. The composition as claimed in any one of the preceding claims, wherein the nanofibres in each layer form a homogenous or heterogeneous matrix of nanofibres.

23. The composition as claimed in any one of the preceding claims, wherein the nanofibres formed from one polymer or protein are bonded by van der waals forces, to form a layer, and nanofibres formed from a different polymer or protein are bonded together to form another layer.

24. The composition as claimed in any one of the preceding claims, wherein multiple dissolvable polymers and non-dissolvable with multiple actives are layered to form composites.

25. The composition as claimed in any one of the preceding claims, wherein two electrospun polymers with multiple actives are electrospun together to form a single layer of nanofibre co polymer.

26. The composition as claimed in claim 25, wherein therapeutic actives are bonded with at least one of the polymer nanofibres.

27. The composition as claimed in any one of the preceding claims, wherein the non-dissolvable electrospun polymer and top layer mesh are configured to function as a physical barrier to prevent infection.

28. The composition as claimed in any one of the preceding claims, wherein the non-dissolvable polymer contain actives are selected from any one of: thrombin, fibrinogen, ceramids, antibodies, antigens, peptides, alginates, collagen, gelatin, polyphenols like catechins and flavonoids, carotenoids, proteins, caffeine, ketones, fatty acids, glycerides, ceramides, carbohydrates, enzymes, serine proteases generally, Cu+, Mg+, Ca+, metal ions in general, metal-organic frameworks (MOFs), phospholipids, curcumin, terpenes, manuka tri-ketones and/or combinations thereof.

29. The composition as claimed in claims 1 to 27, wherein the dissolvable polymer contain actives are selected from any one of: alginates, phyto-compounds, polyphenols, vascular endothelial growth factors (VEGFs), EDTA, metal chelators, Glycosaminoglycans (GAG's) like hyaluronic acid, curcumin, terpenes, manuka tri-ketones generally, collagen, peptides, terpenes, gelatin, polyphenols like catechins and flavonoids, carotenoids, proteins, caffeine, ketones, fatty acids, glycerides, ceramides, carbohydrates, enzymes, serine proteases generally, cationic nanocylinders, Cu+, Mg+, Ca+, metal ions in general, metal-organic frameworks (MOFs), phospholipids, squalene, monoglycerids and triglycerids and/or combinations thereof.

30. The composition as claimed in any one of the preceding claims, wherein the therapeutic actives bound to the non-dissolvable electrospun polymer function as a sacrificial layer, but release actives.

31. The composition as claimed in any one of the preceding claims, wherein the non-dissolvable electrospun polymer has a cross-sectional diameter of lum to 100 nm.

32. A method of treating a wound by applying at least one bioactive compound to a subject to treat and penetrate the skin of the subject, the method comprising the steps of:

(a) selecting a multilayer matrix nanofibre composition as claimed in claims 1 to 31;

(b) moistening the composition; and

(c) applying the composition as a wound dressing to the skin of the subject.

33. A method of skin treatment of a subject in need thereof by application of the composition as claimed in claims 1 to 31, wherein treatments are selected from promoting skin care or repair; assisting or enhancing wound healing; addressing a microbial infection; treating or preventing inflammation; promoting or enhancing cell proliferation; preserving or improving skin elasticity; preserving or improving skin moisture retention; delivering at least one antioxidant to skin; improving scars; and/or combinations thereof.

34. A use of a composition as claimed in claims 1 to 31, in the manufacture of a medicament for treatments selected from promoting skin care or repair; assisting or enhancing wound healing; addressing a microbial infection; treating or preventing inflammation; promoting or enhancing cell proliferation; preserving or improving skin elasticity; preserving or improving skin moisture retention; delivering at least one antioxidant to skin; and/or combinations thereof.

35. A skincare product comprising the composition as claimed in claims 1 to 31, wherein the product is in the form of: a plaster, a bandage, a patch, a mask and a dressing.

36. A method of producing a composition as claimed in claims 1 to 31 comprising the steps of:

(a) selecting at least one bioactive compound;

(b) selecting at least one nanofibre base material;

(c) mixing the bioactive compound or compounds and the nanofibre base material or materials with water or acid to form an aqueous solution; and

(d) electrospinning the solution to form one or more nanofibres physically and/or chemically bonded with the bioactive compound(s).

37. A method of producing a multilayer matrix composition as claimed in claims 1 to 31 comprising the steps of:

(a) producing a first layer by the steps of:

I. providing an active agent or agents;

II. providing a nanofibre base material;

III. mixing the active agent or agents and the nanofibre base material in a solvent phase to form a solution;

IV. electrospinning the solution to form one or more nanofibres chemically bonded with the bioactive(s) in a first layer;

V. electrospinning the base material into nanofibre;

VI. coating the nanofibre with active agents using spraying coating or dip coating method; and

VII. electrospinning the active agents directly with the polymer solution to form active embedded nanofibre matrix.

(b) forming a second or further layer by separately repeating steps (I) to (IV) to form an additional layer; or

(c) combining the first and subsequent layers to form a multilayer composition.

38. The composition as claimed in any one of claims 1 to 31, wherein the composition comprises at least two layers to three or more layers depending on the wound application.