GB2572566A

GB2572566A - Wound healing

Info

Publication number: GB2572566A
Application number: GB1805447.8A
Authority: GB
Inventors: M El-Sherbiny Ibrahim; H Ali Isra; A Khalil Islam
Original assignee: Zewail City Of Science And Tech
Current assignee: Zewail City Of Science And Tech
Priority date: 2018-04-03
Filing date: 2018-04-03
Publication date: 2019-10-09
Anticipated expiration: 2038-04-03
Also published as: GB2572566B; GB201805447D0

Abstract

A wound healing system comprising nanofiber matrix layers separately carrying a mucoadhesive, such as carbomers (polyacrylic acid), polyethylene oxide, polyacrylates, polyvinyl alcohol, polyalkylene glycol, polycarbophil, polyoxyethlene-polyoxypropylene block copolymers, cellulose derivatives, a drug, such as an antiflammatory, an angiogenesis stimulator or a proliferation promoter, and a moisture indicator, such as methylene blue, acyl auramines or indoyl red. Also discussed is use of the wound healing system in the treatment of wounds, such as a burn, an abrasion, a diabetic ulcer and a mucosal ulcer.

Description

Wound Healing

INTRODUCTION

The present invention relates to a wound healing system comprising a mucoadhesive layer comprising a nanofiber matrix, a drug-containing layer comprising a nanofiber matrix, wherein said matrix comprises at least one drug, and an indicator layer comprising a nanofiber matrix, wherein said matrix comprises a moisture indicator. The invention also relates to a method of making the wound healing system and to medical uses of the wound healing system for the treatment of wounds such as diabetic ulcers.

BACKGROUND

Skin is a body organ that protects internal organs. The integrity of the skin is, however, sometimes compromised, for example, as a result of an injury or burn. Skin grafting is the common intervention especially for large skin loss.

Wound healing is a complex multi-step process which involves the reestablishment of dermal and epidermal tissues by various cellular and biochemical processes. These processes include a cascade of events involving inflammation, migration, proliferation, and maturation phases. The wound healing process begins with hemostasis with various inflammatory cells, such as polymorphonuclear cells and macrophages, which play a key role in the early stage of wound healing. In the next stage, keratinocytes migrate to the wound site to initiate tissue re-epithelization and granulation. The process is also characterized by distinct overlapping events such as remodelling and angiogenesis, which are necessary for restoration of blood flow and oxygen supply to the tissue.

Although skin itself has the natural ability of wound healing, open wounds are a soft target for microorganisms which cause infection at wound sites as well as affecting nearby healthy tissues, which consequently delays the wound healing process. Furthermore, some metabolic disorders like diabetes mellitus, which affects millions of people in the world, can mean that wounds cannot proceed through the normal wound healing process. This is, in fact, a major medical challenge. In the US diabetic ulcers affect 25.8 million people and 26.9% of adults 65 years and older, with an estimated $116 billion in direct medical costs. It is estimated that the lifetime risk of developing a foot ulcer is as high as 25% among diabetic patients.

Diabetic ulcers are the most common foot injury leading to infection, lower extremity amputation, and death. By 2050, the prevalence of diabetes is predicted to increase by two- to three-fold. Thus, the clinical and economical burden of chronic nonhealing ulcers will undoubtedly increase.

Historically, wound dressings were considered to take a passive and protective effect in the wound-healing process. The development of efficient wound dressings is very important for wound repair, and an ideal wound dressing should provide a moist wound environment to the wound area, offer protection from secondary infections, remove wound exudate and promote tissue regeneration.

SUMMARY OF INVENTION

Viewed from a first aspect the present invention provides a wound healing system comprising:

(i) a mucoadhesive layer comprising a nanofiber matrix;

(ii) a first drug-containing layer comprising a nanofiber matrix, wherein said matrix comprises at least one drug; and (iii) an indicator layer comprising a nanofiber matrix, wherein said matrix comprises a moisture indicator.

Viewed from a further aspect the present invention provides a method of making a wound healing system as hereinbefore described, comprising:

(i) preparing a mucoadhesive layer on a release liner, preferably by electrospinning;

(ii) preparing a first drug-containing layer on said mucoadhesive layer, preferably by electrospinning;

(iii) optionally forming a second drug-containing layer on said first-drug containing layer, preferably by electrospinning;

(iv) optionally forming a third drug-containing layer on said second drug-containing layer, preferably by electrospinning;

(v) preparing an indicator layer on said first, or if present second or third, drugcontaining layer, preferably by electrospinning; and (vi) applying a backing layer.

Viewed from a further aspect the present invention provides a wound healing system as hereinbefore defined for use in the treatment of wounds.

Viewed from a further aspect the present invention provides the use of at least a first drug in the manufacture of a wound healing system as hereinbefore described for the treatment of wounds.

Viewed from a further aspect the present invention provides a method of treating a wound in a patient in need thereof, comprising:

(i) placing a wound healing system as hereinbefore defined onto a wound on said patient; and (ii) maintaining said system on said wound for a therapeutically effective period of time.

DEFINITIONS

As used herein the term “wound healing system” refers to any topical system capable of delivering a drug to a wound site on the skin or mucosal tissues and adhering to the skin. The term encompasses wound dressings, wound bandages, patches, plasters and tape.

As used herein the term “layer” refers to a continuous body or film of material. Layers do not have any breaks or interruptions therein. Layers may or may not have a uniform thickness. Layers may or may not be planar.

As used herein the term “nanofiber” refers to a fibre having an average diameter of less than 5000 nm.

As used herein the term “nanofiber matrix” refers to a network or scaffold comprising nanofibers.

As used herein the term “biodegradable” refers to a polymer(s) which will break down in vivo.

As used herein the term “mucoadhesive polymer” refers to a polymer that comprises groups that interact with the skin surface. Many mucoadhesive polymers, for example, comprise charged groups that serve to retain the polymer at the wound site.

As used herein the term “carbomer” refers to a polymer comprising acrylic acid and/or methacrylic acid monomers. Carbomers may be homopolymers or copolymers. Optionally carbomers are cross-linked.

As used herein the term “poly(meth)acrylate” refers to a polymer comprising acrylate and/or methacrylate monomers. These polymers are also often referred to as acrylic acid ester and methacrylic acid ester polymers.

As used herein the term “drug-containing layer” refers to a layer comprising at least one drug, and optionally other ingredients.

As used herein the term “backing layer” refers to a layer that is a constituent of a system, which in use of the system, is remote to the skin. The backing layer covers the indicator layer and the drug-containing layer(s) and thereby protects them from exposure to the environment.

As used herein the term “release liner” refers to a removable layer of the system that is removed prior to application of the system to skin. The purpose of the release liner is to prevent the system from loss of drug prior to its application to the skin.

As used herein the term “wound healing” refers to the process by which the structure and function of injured or diseased tissue is restored.

DESCRIPTION OF THE INVENTION

The present invention relates to a wound healing system which provides a single-dose therapy for treatment of complicated wounds, such as diabetic ulcers. The system comprises multiple layers, each comprising a nanofiber matrix. Preferably the system comprises a plurality of drug-containing layers. The nanofiber matrix forming each layer provides the system with a high surface area for the impregnation of drugs into the system and provides the system with a scaffold for reepithelization. Advantageously the system of the invention is prepared layer by layer making it easy to incorporate different drugs and/or other agents into the different layers. This enables the system to be prepared with specific layers for achieving different specific functions, e.g. a layer for indicating progress of the healing process and multiple drug-containing layers, each with different drugs, for targeting improvement of different stages of the wound healing process.

The system comprises:

(i) a mucoadhesive layer comprising a nanofiber matrix;

(ii) a first drug-containing layer comprising a nanofiber matrix, wherein said matrix comprises at least one drug; and (iii) an indicator layer comprising a nanofiber matrix, wherein said matrix comprises a moisture indicator. In use the mucoadhesive layer is in contact with the skin, the indicator layer is remote to the skin and the drug-containing layer is in between the mucoadhesive layer and the indicator layer.

In preferred systems of the present invention the mucoadhesive layer comprises a nanofiber matrix comprising nanofibers having an average diameter of 50 to 4000 nm. Preferably the nanofibers forming the matrix have an average diameter of 60-3950 nm, more preferably 100 to 1000 nm, yet more preferably 150 to 800 nm and still more preferably 150 to 750 nm. Preferably the average diameter of the nanofibers is measured by microscopy, e.g. as described herein in the examples section.

In preferred systems of the present invention the mucoadhesive layer comprises a nanofiber matrix comprising, and more preferably consisting of, nanofibers comprising a mucoadhesive polymer. This facilitates the retention of the system on the skin at the site it is administered and increases its retention time. Suitable biocompatible, mucoadhesive polymers are commercially available.

Preferably the mucoadhesive polymer comprises at least one monomer having -COOH side groups. Such polymers are advantageous as the carboxylic acid side groups act as a neutralising agent for alkaline wounds, e.g. diabetic ulcers, thereby improving the healing process. Particularly preferably the polymer comprises a monomer selected from acrylic acid, methacrylic acid and mixtures thereof.

Preferably the mucoadhesive polymer is selected from carbomer, polyethylene oxide, polyacrylate, polyvinyl alcohol, polyalkylene glycol, polycarbophil, polyoxyethlene-polyoxypropylene block copolymers, cellulose derivatives, natural polysaccharides, chitosan, gelatin, hyaluronic acid, alginitic acid and mixtures thereof. More preferably the mucoadhesive polymer is selected from carbomer, polyethylene oxide and mixtures thereof. Still more preferably the mucoadhesive polymer is a mixture of carbomer and polyethylene oxide.

Different types of carbomer are commercially available. Preferred carbomers for the mucoadhesive layer may be homopolymers or copolymers, but are preferably homopolymers. Particularly preferred carbomers are cross-linked. Representative examples of suitable carbomers for use as the mucoadhesive polymer include those known by the tradename Carbopol®. A preferred Carbopol is Carbopol® 971.

Different types of polyethylene oxide are commercially available. Preferably the polyethylene oxide for the mucoadhesive layer is a homopolymer. Particularly preferably the polyethylene oxide has a weight average molecular weight of 200,000 to 2,000,000, more preferably 500,000 to 1,500,000 and still more preferably 700,000 to 1,000,000.

Preferably the mucoadhesive polymer is a 10:90 to 90:10 (by weight), more preferably 25:75 to 75:25 (by weight), still more preferably 40:60 to 60:40 (by weight) mixture of carbomer and polyethylene oxide. More preferably the mucoadhesive polymer is a 10:90 to 90:10 (by weight), more preferably 25:75 to 75:25 (by weight), still more preferably 40:60 to 60:40 (by weight) mixture of Carbopol® 971 and polyethylene oxide having a weight average molecular weight of 700,000 to 1,000,000.

In preferred systems of the present invention the mucoadhesive layer comprises, e.g. consists of, a nanofiber matrix comprising a mucoadhesive polymer, wherein said matrix comprises an antimicrobial agent. Preferably the anti-microbial agent is encapsulated in particles. Still more preferably the anti-microbial-containing particles are impregnated or encapsulated within the nanofiber matrix. The high surface area of the nanofiber matrix means that relatively high amounts of antimicrobial agent can readily be incorporated into the matrix.

In particularly preferred systems of the present invention the anti-microbialcontaining particles are hyalurosomes. Thus preferably the anti-microbial-containing particles comprise hyaluronic acid and lecithin. An advantage of using hyalurosomes is that the particles only release the anti-microbial agent if they degrade due to contact with hyaluronidase enzyme which is secreted by many microorganisms prevalent in infected wounds.

In preferred systems of the invention the anti-microbial-containing particles are nanoparticles. Preferably the nanoparticles have an average diameter of 30-450 nm, more preferably 50 to 300 nm and still more preferably 60 to 150 nm. The use of nanoparticles advantageously enables a relatively large amount of antimicrobial agent to be incorporated into the system of the invention. Nanoparticles may be prepared by conventional methods known in the art, e.g. nanoprecipitation.

Preferred anti-microbial agents present in the systems of the present invention are selected from moxifloxacin, natamycin, azythromycin, mupirocin, erythromycin, ciprofloxacin, netilmycin, besifloxacin, gatifloxacin, gentamycin sulfate, levofloxacin, ofloxacin, sulfacetamide sodium, tobramycin, bacitracin zinc, Polymyxin B sulfate, neomycin, and neomycin sulfate, acyclovir, valacyclovir, famciclovir, itraconazole, posaconazole, voriconazole, silver sulfadiazine. Particularly preferred anti-microbial agents are selected from levofloxacin, natamycin, ciprofloxacin, tobramycin and polymyxin. An especially preferred anti-microbial agent is ciprofloxacin.

In preferred systems of the invention, the mucoadhesive layer comprising a nanofiber matrix comprises 2-45 wt% of anti-microbial agent, more preferably 5 to 35 wt% of anti-microbial agent and still more preferably 5 to 20 wt% of anti-microbial agent, based on the total weight of the mucoadhesive layer. When the anti-microbial agent is present in anti-microbial-containing particles, the mucoadhesive layer comprising a nanofiber matrix comprises 2-50 wt% of anti-microbial-containing particles, more preferably 5 to 40 wt% of anti-microbial-containing particles and still more preferably 6 to 25 wt% of anti-microbial-containing particles, based on the total weight of the mucoadhesive layer.

In preferred systems of the present invention, the mucoadhesive layer does not comprise any other active agent in addition to the above-mentioned anti-microbial agent. In other words, the anti-microbial agent is preferably the sole active agent present in the mucoadhesive layer.

In preferred systems of the present invention, the mucoadhesive layer has a thickness of 250-2000 μίτι, more preferably 300-1800 μίτι and still more preferably 3001500 μητ

In preferred systems of the present invention, the mucoadhesive layer is electropsun.

In preferred systems of the invention, the indicator layer comprises nanofibers having an average diameter of 50 to 4000 nm. Preferably the nanofibers forming the matrix have an average diameter of 60-3950 nm, more preferably 100 to 1000 nm, yet more preferably 150 to 800 nm and still more preferably 150 to 750 nm. Preferably the average diameter of the nanofibers is measured by microscopy, e.g. as described herein in the examples section.

In preferred systems of the invention the indicator layer comprises a nanofiber matrix comprising, and more preferably consisting of, a polymer selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid, poly-D-lactic acid, poly-L-lactic acid, PLGA-dimethacrylate, fluorescent PLGA polymers, a poly(meth)acrylate, a poly(methyl)methacrylate, a poly(hydroxymethyl)methacrylate, a polyanhydride, a polyorthoester, a polyetherester, a polycaprolactone, a polysaccharide, a polyester, a polydioxanone, a polygluconate, an ethyl cellulose, cellulose derivatives, chitosan derivatives or mixtures or combinations thereof. More preferably the indicator layer comprises a nanofiber matrix comprising, e.g. consisting of, a polymer selected from polylactic acid, poly-D-lactic acid, poly-L-lactic acid or mixtures thereof. Still more preferably the indicator layer comprises a nanofiber matrix comprising or consisting of poly-L-lactic acid.

Suitable polymers, e.g. polylactic acid, for use in the nanofiber matrix of the indicator layer are commercially available. Suitable polylactic acid polymers are commercially available under the trade designation INGEO (e.g., INGEO 4032D, INGEO 4043D, and INGEO 4060D) from NatureWorks

In systems of the present invention the indicator layer comprises a moisture indicator. The moisture indicator is highly beneficial as it can provide a measure of the progress of the healing process hidden from sight, under the system. This is because water is released as the healing process progresses. In preferred systems of the invention the moisture indicator is selected from methylene blue, acyl auramines, acylleucophenothiazines, alpha- and beta-unsaturated aryl ketones, azaphthalides, basic mono azo dyes, 10-benzoyl-N,N,N',N'-tetraethyl-3,7-diamino-10H-phenoxazine, chromogenic azaphthalide compounds, diaryl phthalides, diphenylmethanes, dithiooxamide, di[bis-(indolyl)ethylenyl]tetrahalophthalides, fluoran derivatives (3dialkylamino-7-dialkylamylfluoran),3-(indol-3-yl)-3-(4-substituted aminophenyl) phthalides, bis-(indolyl)ethylenes, indolyl red, leucoauramines, 3-methyl-2,2spirobi(benzo-[f]-chromene), phenoxazine, phthalides including crystal violet lactone, malachite green lactone, phthalide red, phthalide violet, phthalans, benzoindolinospiropyrans, rhodamine beta lactams, spiropyrans, triphenylmethanes including gentian violet and malachite green. In preferred systems of the invention the moisture indicator is methylene blue.

In preferred systems of the invention the indicator layer consists of a nanofiber matrix comprising an indicator. Preferably the indicator layer does not comprise any drug.

In preferred systems of the present invention, the indicator layer has a thickness of 50-4000 qm, more preferably 60-3950 qm and still more preferably 503800 μητ

In preferred systems of the present invention, the indicator layer is electropsun.

In preferred systems of the invention the first drug-containing layer comprises nanofibers having an average diameter of 50 to 4000 nm. Preferably the nanofibers forming the matrix have an average diameter of 60-3950 nm, more preferably 100 to 1000 nm, yet more preferably 150 to 800 nm and still more preferably 150 to 750 nm. Preferably the average diameter of the nanofibers is measured by microscopy, e.g. as described herein in the examples section.

In preferred systems of the invention the first drug-containing layer comprises a nanofiber matrix comprising, and more preferably consisting of, a polymer selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid, poly-D-lactic acid, poly-L-lactic acid, PLGA-dimethacrylate, fluorescent PLGA polymers, a poly(meth)acrylate, a poly(methyl)methacrylate, a poly(hydroxymethyl)methacrylate, a polyanhydride, a polyorthoester, a polyetherester, a polycaprolactone, a polysaccharide, a polyester, a polydioxanone, a polygluconate, an ethyl cellulose, cellulose derivatives, chitosan derivatives or mixtures or combinations thereof. More preferably the first drugcontaining layer comprises a nanofiber matrix comprising, e.g. consisting of, a polymer selected from polylactic acid, poly-D-lactic acid, poly-L-lactic acid or mixtures thereof. Still more preferably the first drug-containing layer comprises a nanofiber matrix comprising, e.g. consisting of, poly-L-lactic acid.

Suitable polymers, e.g. polylactic acid, for use in the nanofiber matrix of the indicator layer are commercially available. Suitable polylactic acid polymers are commercially available under the trade designation INGEO (e.g., INGEO 4032D, INGEO 4043D, and INGEO 4060D) from NatureWorks.

In preferred systems of the invention the first-drug containing layer comprises a nanofiber matrix comprising, e.g. consisting of, nanofibers comprising a drug. Preferably the drug is encapsulated or impregnated by the nanofiber matrix.

In preferred systems of the invention the first drug-containing layer comprises a nanofiber matrix comprising a drug selected from an anti-inflammatory, an angiogenesis stimulator, a proliferation promoter or mixtures thereof. When only a single drug-containing layer is present in the system of the invention, preferably the drug-containing layer comprises each of an anti-inflammatory drug, an angiogenesis stimulator drug and a proliferation promoter drug. When two drug-containing layers are present in the system of the invention, preferably the first drug-containing layer comprises a proliferation promoter drug and optionally an angiogenesis stimulator drug. When at least three, e.g. three, drug-containing layers are present in the system of the invention, preferably the first drug-containing layer comprises a proliferation promoter drug.

Preferred anti-inflammatory drugs present in the systems of the present invention are selected from nitric oxide, atorvastatin, fluvastatin, lovastatin, pravastatin, pitavastatin, rosuvastatin, simvastatin, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, nifedipine, oxaprozin, piroxicam, sulindac, prednisolone, dexamethasone, beclomethasone, budesonide and hydrocortisone.

Preferred angiogenesis stimulator drugs present in the systems of the present invention are selected from nitric oxide, Icariin, Sildenafil, Udenafil, Mirodenafil, Tadalafil, Acetildenafil, Aildenafil, Vardenafil, Lodenafil, Sulfoaildenafil, Avanafil, Zaprinast, diltiazem, glyceryl trinitrate, Isosorbide dinitrate, and isosorbide mononitrate.

Preferred proliferation promoter drugs present in the systems of the present invention are selected from phenytoin, nitric oxide, pentoxifylline, and retinoids.

In preferred systems of the invention, the first drug-containing layer comprising a nanofiber matrix comprises 2 to 45 wt% of drug, more preferably 5 to 35 wt% of drug and still more preferably 5 to 20 wt% of drug, based on the total weight of drug in the layer. In particularly preferred systems of the invention, wherein the first drugcontaining layer comprising a nanofiber matrix only comprises one type of drug, and preferably a proliferation promoter drug, the first drug-containing layer comprising a nanofiber matrix comprises 1 to 40 wt% of drug, more preferably 2 to 30 wt% of drug and still more preferably 5 to 20 wt% of drug, based on the total weight of drug in the layer.

In preferred systems of the present invention, the first drug-containing layer has a thickness of 250-2000 qm, more preferably 300-1800 μΐη and still more preferably 300-1500 μΓΠ.

In preferred systems of the present invention, the first drug-containing layer is electropsun.

Preferred systems of the present invention further comprise a second drugcontaining layer comprising a nanofiber matrix. In preferred systems of the invention the second drug-containing layer comprises nanofibers having an average diameter of 50 to 4000 nm. Preferably the nanofibers forming the matrix have an average diameter of 60-3950 nm, more preferably 100 to 1000 nm, yet more preferably 150 to 800 nm and still more preferably 150 to 750 nm. Preferably the average diameter of the nanofibers is measured by microscopy, e.g. as described herein in the examples section.

In preferred systems of the invention the second drug-containing layer comprises a nanofiber matrix comprising, and more preferably consisting of, a polymer selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid, poly-D-lactic acid, poly-L-lactic acid, PLGA-dimethacrylate, fluorescent PLGA polymers, a poly(meth)acrylate, a poly(methyl)methacrylate, a poly(hydroxymethyl)methacrylate, a polyanhydride, a polyorthoester, a polyetherester, a polycaprolactone, a polysaccharide, a polyester, a polydioxanone, a polygluconate, an ethyl cellulose, cellulose derivatives, chitosan derivatives or mixtures or combinations thereof. More preferably the second drug-containing layer comprises a nanofiber matrix comprising, e.g. consisting of, a polymer selected from polylactic acid, poly-D-lactic acid, poly-Llactic acid or mixtures thereof. Still more preferably the second drug-containing layer comprises, e.g. consists of, a nanofiber matrix comprising poly-L-lactic acid. Preferably the first and second drug-containing layers comprise a nanofiber matrix comprising the same polymer and in particular poly-L-lactic acid.

In preferred systems of the invention the second drug-containing layer comprises, and more preferably consists of, a nanofiber matrix comprising nanofibers comprising a drug. Preferably the drug is encapsulated by, or impregnated in, the nanofiber matrix.

In preferred systems of the invention the second drug-containing layer comprises a nanofiber matrix comprising a drug selected from an anti-inflammatory, an angiogenesis stimulator, a proliferation promoter or mixtures thereof. When only a first and second drug-containing layer is present in the system of the invention, preferably the first drug-containing layer comprises a proliferation promoter drug and an angiogenesis stimulator drug and the second drug-containing layer comprises an antiinflammatory drug or the first drug-containing layer comprises a proliferation promoter drug and the second drug-containing layer comprises an angiogenesis stimulator drug and an anti-inflammatory drug. When at least three, e.g. three, drug-containing layers are present in the system of the invention, preferably the second drug-containing layer comprises an angiogenesis stimulator drug.

In systems of the present invention comprising first and second drug-containing layers, preferably the first drug-containing layer is present on top of the mucoadhesive layer and the second drug-containing layer is present on top of the first drug-containing layer. Preferably the order of the layers is: (1) mucoadhesive layer; (2) first drugcontaining layer; (3) second drug-containing layer; and (4) indicator layer. In use the mucoadhesive layer is in contact with the skin of the patient.

In preferred systems of the invention, the second drug-containing layer comprising a nanofiber matrix comprises 2 to 45 wt% of drug, more preferably 5 to 35 wt% of drug and still more preferably 5 to 20 wt% of drug, based on the total weight of drug in the layer. In particularly preferred systems of the invention, wherein the second drug-containing layer comprising a nanofiber matrix only comprises one type of drug, and preferably a angiogenesis stimulator drug, the second drug-containing layer comprising a nanofiber matrix comprises 1 to 40 wt% of drug, more preferably 2 to 30 wt% of drug and still more preferably 1 to 20 wt% of drug, based on the total weight of drug in the layer.

In preferred systems of the present invention, the second drug-containing layer has a thickness of 250-2000 pm, more preferably 300-1800 pm and still more preferably 300-1500 pm.

In preferred systems of the present invention, the second drug-containing layer is electropsun.

Preferred systems of the present invention, further comprise a third drugcontaining layer comprising a nanofiber matrix. In preferred systems of the invention the third drug-containing layer comprises nanofibers having an average diameter of 50 to 4000 nm. Preferably the nanofibers forming the matrix have an average diameter of 60-3950 nm, more preferably 100 to 1000 nm, yet more preferably 150 to 800 nm and still more preferably 150 to 750 nm. Preferably the average diameter of the nanofibers is measured by microscopy, e.g. as described herein in the examples section.

In preferred systems of the invention the third drug-containing layer comprises, and more preferably consists of, a nanofiber matrix comprising a polymer selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid, poly-D-lactic acid, poly-L-lactic acid, PLGA-dimethacrylate, fluorescent PLGA polymers, a poly(meth)acrylate, a poly(methyl)methacrylate, a poly(hydroxymethyl)methacrylate, a polyanhydride, a polyorthoester, a polyetherester, a polycaprolactone, a polysaccharide, a polyester, a polydioxanone, a polygluconate, an ethyl cellulose, cellulose derivatives, chitosan derivatives or mixtures or combinations thereof. More preferably the third drugcontaining layer comprises a nanofiber matrix comprising, e.g. consisting of, a polymer selected from polylactic acid, poly-D-lactic acid, poly-L-lactic acid or mixtures thereof. Still more preferably the third drug-containing layer comprises a nanofiber matrix comprising, e.g. consisting of, poly-L-lactic acid.

Suitable polymers, e.g. polylactic acid, for use in the nanofiber matrix of the third drug-containing layer are commercially available. Suitable polylactic acid polymers are commercially available under the trade designation INGEO (e.g., INGEO 4032D, INGEO 4043D, and INGEO 4060D) from NatureWorks.

Preferably the first, second and third drug-containing layers comprise a nanofiber matrix comprising the same polymer. Preferably the drug-containing layer in contact with the indicator layer and the indicator layer comprise a nanofiber matrix comprising the same polymer. Preferably the first, second and third drug-containing layers and the indicator layer comprise a nanofiber matrix comprising the same polymer.

In preferred systems of the invention the third drug-containing layer comprises a nanofiber matrix comprising, and more preferably consisting of, nanofibers comprising a drug. Preferably the drug is encapsulated by, or impregnated in, the nanofiber matrix.

In preferred systems of the invention the third drug-containing layer comprises a nanofiber matrix comprising a drug selected from an anti-inflammatory, an angiogenesis stimulator, a proliferation promoter or mixtures thereof. When at least three, e.g. three, drug-containing layers are present in the system of the invention, preferably the first drug-containing layer comprises a proliferation promoter drug, the second drug-containing layer comprises an angiogenesis stimulator drug and the third drug-containing layer comprises an anti-inflammatory drug.

In systems of the present invention comprising first, second and third drugcontaining layers, preferably the first drug-containing layer is present on top of the mucoadhesive layer, the second drug-containing layer is present on top of the first drug-containing layer and the third drug-containing layer is present on top of the second drug-containing layer. Preferably the order of the layers is: (1) mucoadhesive layer; (2) first drug-containing layer; (3) second drug-containing layer; (4) third-drugcontaining layer and (5) indicator layer. In use the mucoadhesive layer is in contact with the skin of the patient.

In preferred systems of the invention, the third drug-containing layer comprising a nanofiber matrix comprises 2 to 45 wt% of drug, more preferably 5 to 35 wt% of drug and still more preferably 5 to 20 wt% of drug, based on the total weight of drug in the layer. In particularly preferred systems of the invention, wherein the third drugcontaining layer comprising a nanofiber matrix only comprises one type of drug, and preferably an anti-inflammatory drug, the third drug-containing layer comprising a nanofiber matrix comprises 1 to 40 wt% of drug, more preferably 2 to 30 wt% of drug and still more preferably 5 to 20 wt% of drug, based on the total weight of drug in the layer.

In preferred systems of the present invention, the third drug-containing layer has a thickness of 250-2000 qm, more preferably 300-1800 qm and still more preferably 300-1500 μητ

In preferred systems of the present invention, the third drug-containing layer is electropsun.

In preferred systems of the invention each of the drug-containing layers present therein is electrospun. In still further preferred systems of the invention each of the layers present therein in use, other than the backing layer, is electrospun. An advantage of employing electrospun nanofiber matrices in the system of the invention is that it is porous, allows for cell migration there through, and presents a large surface area as a scaffold for tissue re-epithelization and granulation. This improves the wound healing process.

The drug-containing layers of the present invention may optionally comprise a release rate controlling agent. Thus, in some systems of the present invention one or more of the drug-containing layers further comprises a rate controlling agent. The rate controlling agent may increase or decrease the rate of release of drug from the layer. Examples of rate controlling agents that increase the rate of release of drug include lecithin, phosphatidylcholine, polyethylene oxide)-poly (propylene oxide)poly(ethylene oxide), polysorbate, sorbitan monooleate, oleoyl polyoxyl-6 glycerides, polyetholylated stearyl alcohol, and polyethoxylated castor oil. Examples of rate controlling agents that decrease the rate of release of drug include γ-tocopherol, cholesterol, glyceryl Behenate, and glyceryl palmitostearate.

Particularly preferred systems of the invention comprise a first and a second drug-containing layer, wherein the order of the layers in said system is:

(a) mucoadhesive layer;

(b) first drug-containing layer;

(c) second drug-containing layer; and (d) indicator layer.

In such systems the first drug-containing layer preferably comprises a nanofiber matrix comprising a proliferation promoter and optionally an angiogenesis stimulator and the second drug-containing layer comprises a nanofiber matrix comprising an antiinflammatory drug or the first drug-containing layer preferably comprises a nanofiber matrix comprising a proliferation promoter and the second drug-containing layer comprises a nanofiber matrix comprising an angiogenesis stimulator and an antiinflammatory drug.

In yet further preferred systems of the invention comprising first, second and third drug-containing layers, the order of the layers in said system is:

(a) mucoadhesive layer;

(b) first drug-containing layer;

(c) second drug-containing layer;

(d) third drug-containing layer; and (d) indicator layer.

In such systems the first drug-containing layer preferably comprises a nanofiber matrix comprising a proliferation promoter, the second drug-containing layer preferably comprises a nanofiber matrix comprising an angiogenesis stimulator and the third drugcontaining layer comprises a nanofiber matrix comprising an anti-inflammatory drug.

Preferred drugs present in the systems of the present invention are selected from nitric oxide, atorvastatin, fluvastatin, lovastatin, pravastatin, pitavastatin, rosuvastatin, simvastatin, aspirin, celecoxib, diclofenac, diflunisal, etodolac, ibuprofen, indomethacin, ketoprofen, ketorolac, nabumetone, naproxen, nifedipine, oxaprozin, piroxicam, sulindac, prednisolone, dexamethasone, beclomethasone, budesonide, hydrocortisone, Icariin, Sildenafil, Udenafil, Mirodenafil, Tadalafil, Acetildenafil, Aildenafil, Vardenafil, Lodenafil, Sulfoaildenafil, Avanafil, Zaprinast, diltiazem, glyceryl trinitrate, Isosorbide dinitrate, isosorbide mononitrate, phenytoin, pentoxifylline, and retinoids.

In preferred systems of the present invention the total amount of drug is 2 to 45 wt%, more preferably 5 to 35 wt% and still more preferably 5 to 20 wt%, based on the total weight of the system. In further preferred systems of the present invention the total weight of the nanofiber matrix is 55 to 98 wt%, more preferably 65 to 95 wt% and still more preferably 80 to 95 wt% of the total weight of the system.

Each of the layers of the system of the present invention may further comprise other conventional excipients, e.g. tackifiers, pH regulators, fillers, softeners, antioxidants, and viscosity modifying agents. Such additional excipients are preferably added in an amount of less than 30 %wt, more preferably less than 20 %wt and even more preferably less than 10 %wt based on the total weight of the layer.

Preferred systems of the invention further comprise a release liner which is removable or detachable. When present, the release liner is present on the mucoadhesive layer. The release liner is removed or detached prior to use of the system to expose a surface thereof for contact with the skin. Preferred systems of the present invention are self-adhering. Thus, when the release liner is removed and the system is applied to the patient’s skin, the system remains attached thereto without there being a need for any separate attachment mechanism, e.g. straps or ties.

Representative examples of release liners include polyolefin (e.g. high and low density polyethylene, polypropylene), fluoropolymer (e.g. polytetrafluoroethylene), nylon, cellulose derivatives, ethylene-vinyl acetate, vinyl acetate, polyvinylchloride, polyurethane, polyesters (e.g. polyethylene phthalate, polyethylene terephthalate, polybutylene terephthalate or polyethylene naphthalate) and laminates of the aforegoing. Preferably the release liner comprises silicone, fluropolymer or a mixture thereof. Suitable release liners are commercially available form a range of suppliers.

Preferred systems of the invention further comprise a backing layer. The backing layer is preferably impermeable to any drug present in the system. Preferably the backing layer is occlusive. The backing layer preferably serves as a protective cover and may also provide a support function. Preferably the backing layer is flexible so that it can accommodate movement of the patient without breaking. Preferably the backing layer is transparent so the underlying indicator layer can be seen.

The backing layer may be formed from a range of different materials including film, fabric, foamed sheet, microporous sheet, textile fabrics, foil or a laminate of the afore-going. Examples of backing layers comprise a polyolefin (e.g. high and low density polyethylene, polypropylene), fluoropolymer (e.g. polytetrafluoroethylene), nylon, cellulose derivatives, ethylene-vinyl acetate, vinyl acetate, polyvinylchloride, polyurethane, polyesters (e.g. polyethylene phthalate, polyethylene terephthalate, polybutylene terephthalate or polyethylene naphthalate), metal foils (e.g. aluminium) and laminates of the afore-going. Suitable backing layers are available from a range of commercial suppliers.

Preferred systems of the present invention have a total thickness of 0.5 to 10 mm, more preferably 1-7.5 mm and still more preferably 2-6 mm. An advantage of the system of the present invention is that it is constructed layer by layer meaning that systems of variable thickness can readily be prepared.

Preferably the cross section of the system of the invention is circular, square or rectangular. Preferably the system, e.g. dressing or bandage, is sized and shaped to readily fit onto the wound site, or a part thereof. Optionally the system is provided in the form of a strip or roll which can be cut to a suitable size for use. Preferably the system has a surface area of 5-2000 cm², more preferably 10-1500 cm² and still more preferably 20-1200 cm², e.g. when in use.

The system of the present invention is preferably in the form of a wound dressing, wound bandage, patch, plaster or tape. Particularly preferably the system of the invention is a wound bandage or wound dressing. Preferably the system is solely formed from nanofiber matrix layers, and particularly electrospun nanofiber matrix layers, and optionally a backing layer and a release layer. Preferably the system of the invention does not comprise any additional layers.

The system of the present invention may comprise 3, 4, 5, 6, 7 or more layers. Preferred systems comprise at least 5 layers and especially preferably 7 layers.

In preferred systems of the present invention, each of the layers present therein is preferably planar. Preferably each of the drug-containing layers is continuous. Particularly preferably the drug-containing layers do not comprise any channels.

Preferably the system of the present invention has an in vitro biodegradability in PBS, e.g. as measured according to the method described in the examples for 30 days, of at least 5 %wt and more preferably at least 10 %wt. Thus in preferred systems of the present invention, less than 90 %wt and preferably less than 85 %wt of the system remains after exposure to PBS for 30 days. This level of in vitro biodegradability indicates that the system is well suited for medical use whilst having the required level of durability.

Preferably the system of the present invention achieves its maximum swelling in PBS within 2 hour of being placed in the PBS, e.g. as determined according to the method in the examples. Preferably the system of the present invention achieves a swelling of at least 50 %wt, and more preferably at least 300 %wt in PBS within 2 hours of being placed in the PBS, e.g. as determined according to the method in the examples.

An advantage of the system of the present invention is that a relatively high total amount of drug may be present therein. This is because of the combined effect of the high surface area of the nanofibers and the multiple drug-containing layers. Advantageously a combination of different drugs can be released in a controlled manner. As a result, and despite the relatively small size of the systems of the present invention, a system preferably comprises an amount of drug(s) that is required for a complete treatment regime. In other words, the system provides a single dose treatment regimen.

A further advantage of the systems of the present invention is that the system provides controlled-release of drug(s) over a period of at least 2 days, more preferably at least 3 days and still more preferably at least 7 days. Preferably the system provides controlled-release of drug(s) over a period of 2 to 35 days, more preferably 2 to 22 days and still more preferably 3-21 days. An advantage of the system of the present invention is that the drug release rate may be modified by selection of the polymers used to form the nanofiber matrices present in the layers and/or the incorporation of rate controlling agents in the layers and/or by the selection of the drug(s).

The present invention also relates to a method of making a wound healing system as hereinbefore described, comprising:

In a preferred method of the invention the mucoadhesive layer is prepared on a release liner by electrospinning. This is a well-known technique and the skilled person would readily understand what is involved. Preferably a solution of the mucoadhesive polymer is prepared and then electrospun. Optionally a solution or dispersion of the anti-microbial agent is mixed with the polymer solution prior to electrospinning. Preferably the solvent is water, ethanol or a mixture thereof. The conditions employed during electrospinning preferably comprise at least one, preferably two, still more preferably all, of the following conditions:

Flow rate: 0.5-5 ml/hour, preferably 1.5-2.5 ml/hr;

Applied voltage: 10-50 kV, preferably 15-25 kV;

Distance between needle tip and receiving collector: 5-30 cm, preferably 10 to cm;

Speed: 50-250 mm/sec, preferably 100 to 200 mm/sec;

Temperature inside electrospinner: 20-40 °C, preferably 25-35 °C; and

Humidity inside electrospinner: 20-70 %, preferably 40-50 %.

In a preferred method of the invention the indicator layer is prepared by electrospinning. Preferably a solution of a polymer as hereinbefore described and an indicator is prepared and then electrospun. Preferably the solvent is DCM.

In a preferred method of the invention the drug-containing layer(s) is prepared by electrospinning. Preferably a solution of a polymer as hereinbefore described and a drug(s) is prepared and then electrospun. Preferably the solvent is DCM.

The conditions employed during electrospinning of the indicator and drugcontaining layers preferably comprise at least one, preferably two, still more preferably all, of the following conditions:

Flow rate: 0.5-5 ml/hour, preferably 1.5-2.5 ml/hr;

Applied voltage: 10-50 kV, preferably 15-25 kV;

Speed: 50-250 mm/sec, preferably 100 to 200 mm/sec;

Temperature inside electrospinner: 20-40 °C, preferably 25-35 °C; and

Humidity inside electrospinner: 20-70 %, preferably 40-50 %.

The electrospinning process yields polymer fibers that create a matrix that is used to form the system of the invention. If necessary, the matrix produced by electrospinning may be cut to the desired shape and/or size.

The system of the present invention is used for the treatment of wounds including acute wounds and chronic wounds. Alternatively the present invention provides use of at least a first drug in the manufacture of a wound healing system as hereinbefore described for the treatment of wounds. In a further alternative the present invention provides a method of treating a wound in a patient in need thereof, comprising:

(i) placing a wound healing system as hereinbefore described onto a wound on the patient; and (ii) maintaining said system on said wound for a therapeutically effective period of time.

Representative examples of wounds that may be treated include a bum, an abrasion, a laceration, a puncture, an avulsion, an incision, a graft, a lesion caused by an infectious agent, a chronic venous ulcer, a diabetic ulcer, a compression or decubitus ulcer and a mucosal ulcer. The systems of the invention are particularly suitable for the treatment of diabetic ulcers and especially diabetic foot ulcers.

DESCRIPTION OF THE FIGURES

Figure 1 is a schematic of a cross section of a wound healing system of the present invention;

Figure 2 is a schematic of a cross section of a preferred wound healing system of the present invention;

Figure 3a shows a SEM image of PLI_A nanofibers electrospun from chloroform solution;

Figure 3b shows a SEM image of PLLA nanofibers electrospun from a DCM:DMF (1:1) solution;

Figure 3c shows a higher magnification image of nanofibers electrospun from a DCM solution to show their highly porous surface;

Figure 3d shows a still higher magnification image of nanofibers electrospun from a DCM solution to show their highly porous surface;

Figure 4a) shows a SEM image of electrospun carbapol nanofibers and showing beading;

Figure 4b) shows a SEM image of electrospun PEO nanofibers and showing beading;

Figure 4c) shows a SEM image of electrospun nanofibers with carbapol: PEO in a ratio of 75:25;

Figure 4d) shows a SEM image of electrospun nanofibers with carbapol: PEO in a ratio of 50:50;

Figure 4e) shows a SEM image of electrospun nanofibers with carbapol: PEO in a ratio of 25:75;

Figure 5a is a plot of wavelength of maximum absorbance (nm) versus time (minutes) for each different humidity conditions tested;

Figure 5b is a photograph of a dry system (left hand side) and a system exposed to high humidity for an extended period of time (right hand side);

Figure 6 is a plot of biodegradability (weight remaining %) versus time (days) for the different layers present in a system;

Figure 7 is a plot of swelling (%) versus time (hours) for the different layers present in a system;

Figure 8a is a plot of phenytoin released (%) versus time (days) from nanofiber layers with different compositions;

Figure 8b is a plot of sildenafil released (%) versus time (days) from nanofiber layers with different compositions;

Figure 9 is a bar chart of optical density of fibroblasts (cell viability) for the different types of nanofiber layers present in the system of the invention;

Figure 10 is a SEM image of cells attached to a surface of the system of the invention; and

Figure 11 shows microscope images for skin specimens taken from each animal test group overtime (days).

DETAILED DESCRIPTION OF THE FIGURES

Referring to Figure 1, it shows a cross section of a system of the present invention that is ready to be placed on the skin of a patient. The system 1 is a wound dressing and comprises three nanofiber matrix layers and a backing layer. The bottom layer 2, which, in use, is placed on the skin, is a mucoadhesive layer comprising a nanofiber matrix. The second layer 3 is the drug layer. It comprises a nanofiber matrix comprising at least one drug. The third layer 4, is an indicator layer comprising a nanofiber matrix comprising a moisture indicator, e.g. methylene blue. The indicator changes colour from purple to blue when moisture is released due to wound healing. The top layer is a backing layer which prevents moisture from entering the dressing from the outside environment. The top layer is transparent to enable the indicator layer to be seen.

Referring to Figure 2, it shows a cross section of a preferred system 1 of the present invention. In Figure 2 the system is in its packaged form. The system comprises 7 layers as follows: layer 10 is release liner, layer 11 is a mucoadhesive layer comprising a nanofiber matrix, wherein said matrix comprises an anti-microbial agent, layer 12 is a first drug-containing layer comprising a nanofiber matrix, wherein said matrix comprises an proliferation promoter drug, layer 13 is a second drug containing layer comprising a nanofiber matrix, wherein said matrix comprises an angiogenesis stimulator drug, layer 14 is a third drug containing layer comprising a nanofiber matrix, wherein said matrix comprises an anti-inflammatory drug, layer 15 is an indicator layer comprising a nanofiber matrix, wherein said matrix comprises a moisture indicator and layer 16 is a backing layer. Preferably each of layers 11-14 is electrospun.

The advantages of the system of the present invention over woven wound dressings include:

1. High porosity with swelling capability for hemostasis, absorbing excess wound exudates and allowing oxygen and moisture permeability.

2. Ability to neutralize wound pH that is altered by microbial infection.

3. Allow cell migration to the wound site that is essential to initiate tissue re-epithelization and granulation.

4. Monitors the healing progress without removing the patch during treatment with the aid of a humidity-responsive sensor.

5. Allow enhanced adhesion to tissue.

6. Confers accurate dose delivery, sustained drug release with constant rate, prolonged drug activity, reduced dose frequency of administration as a singledose therapy.

7. Allows improved drug bioavailability, and low incidence of systemic sideeffects.

8. Promotes natural tissue regeneration and efficient wound healing without scar formation.

9. A single-dose therapy.

10. Demonstrates enhanced antimicrobial activity.

11. Providing a convenient wound remedy to act on all wound healing cascade events involving inflammation, migration, proliferation, and maturation phases beside infection elimination.

12. The high surface area, high porosity and variable pore-size distribution of the developed electrospun nanofibers patch allowed rapid fibroblasts spreading, proliferation, and healing through increasing the oxygen and nutrients diffusion and the high surface area to volume ratio quickly activates the cell signalling pathway.

13. The morphological resemblance of the developed patch to the natural extracellular matrix of the wound supports the fibroblast’s growth in order to repair the damaged tissue.

EXAMPLES

Materials

Polylactic acid (PLLA), Carbapol, Polyethylene oxide (PEO), Pegylated Chitosan, Polyvinyl Pyrrolidone (PVP), PEG 400, Sodium Hyalouronate, Lecithin, Tetramethylorthosilicate (TMOS), Phenytoin, Sildenafil, Simvastatin, Ciprofloxacin, LCysteine, Sodium Nitrite, Sodium Triphosphate (TPP), Lithium Chloride, Potassium Acetate, Magnesium Chloride, Magnesium Nitrate, Sodium Chloride, Potassium Sulphate, Dichloromethane (DCM), Dimethylformamide (DMF), Chloroform (CHCL3), Acetic Acid, Hydrochloric Acid (HCI), Ethanol, Buffer pH 7.4, MTT (3-(4,5dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) kit.

All materials were purchased from commercial suppliers.

Equipment

Electrospinning was carried out using NANON-O1A electrospinner (MECC, Japan).

SEM was carried out under low vacuum using (Nona Nano SEM, FEI, USA).

Absorbance was measured using a UV-Vis spectrophotometer (Evolution UV 600, Thermo Scientific, USA).

Water vapor permeability was tested using ASTM E96 desiccant method.

Porosity of the bandage was measured using a pycnometer (Ultrapyc 1200 e, Quantachrome instruments, USA).

Cell proliferation was assessed using an optical microscope (Olympus).

Blood sugar levels were measured using an Accu-check active glucometer (Alpha Medical, UAE).

Preparation of the Wound Healing bandage

Preparation of Nitric Oxide Releasing Nanoparticles

An acidified amount of tetramethylorthosilicate (2.5 mL/0.6 ml HCI) and water were sonicated for at least 10 minutes in an ice bath and then added dropwise, with stirring, into a previously prepared buffer solution of pegylated chitosan (0.5% w/v) of low molecular weight, sodium nitrite solution (1 g in 30 mM PBS at pH 7.5), L-Cysteine (40 mg/mL), PEG 400 (0.5 mL PEG/10 mL of solution) and PVP (6.25 mg). The solution was left on a stirrer until gelation occurred before being frozen and lyophilized to obtain dry fine powder of NO releasing nanoparticles.

Preparation of PLLA Electrospinning Solution

PLLA (10% w/v) was dissolved in different solvent systems to investigate the effect of the solvent on the properties of the nanofibers produced. Different solvents impact on the porosity of the nanofibers produced depending on the surface tension and the volatility of the solution. Increasing the porosity of the nanofibers will increase the perfusion of oxygen and nutrients within the system and the wound, thus enhancing the proliferation and healing process.

Three different solvent systems were used: a) DCM (100%), b) CHCI₃ (100%) and c) DCM: DMF (1:1). This is considered to be the fundamental electrospinning solution. The solution of each layer will contain this basic solution in addition to certain additives according to the function of each layer as will be discussed in the following subsections. Optionally lecithin may be added to the solution to increase the rate of drug release from the system. Lecithin increases the hydrophilicity and consequently the swellability of the nanofibers which leads to an increase in the rate of the drug release. This can be tailored and manipulated according to the severity of the wound to be treated.

Preparation of the PLLA Indicator Electrospinning Emulsion

An amount of 500 mg of urea and 10 mg of dye (methylene blue) was dissolved in 1 ml of distilled water until compete dissolution, then added dropwise, with vigorous stirring, to an amount of 10 ml of the previously prepared PLLA solution described above until a homogenous emulsion was obtained.

Preparation of Drug Loaded PLLA Electrospinning Solution

An amount of 100 mg of each of Phenytoin, Sildenafil or Simvastatin was added individually to an amount of 10 ml of the previously prepared PLLA solution described above to form the electrospinning solution for each layer of the final system. Specifically the required amounts of Phenytoin and Sildenafil were dissolved individually in the minimum amount of acetone and acetic acid respectively, and then concentrated in the hood before being added to the PLLA solution. The required amount of Simvastatin was dissolved immediately in PLLA solution. The required amount of NO releasing nanoparticles was also dispersed directly into PLLA solution.

Preparation of Carbapol-PEO Electrospinning Solution

Carbapol (3% w/v) and PEO (3% w/v) were dissolved separately in ethanol and distilled water respectively until clear solutions were obtained. The solutions were then mixed in different ratios to investigate the optimum ratio for electrospinning without beading. Five different solutions were prepared which comprised: Carbapol and PEO polymers in the ratios of 100:0, 75:25, 50:50, 25:75 and 0:100.

Preparation of Antibiotic Loaded Hyalorosomes

Hyalorosomes loaded with ciprofloxacin, as a model antibiotic, were prepared using hyaluronic acid and lecithin. After hydration and sonication, the suspension containing antibiotic loaded hyalorosomes was incorporated into the Carbapol-PEO layer (the adhesive layer towards the wound). The antibiotic will be released only as a response to the presence of hyualonaze enzyme which is secreted by many of the microorganisms prevalent in infected wounds.

Fabrication of Electrospun System Layers

Wound bandages, which are an example of a system of the invention, were produced using an electrospinning technique. Different formulations were investigated in order to test the effect of each drug individually and in combinations. The formulations differ in the number of layers and drugs used. They can be manipulated according to the complication, severity and wound site as well as according to each patient.

The outermost layer is comprised of PLLA containing an indicator to facilitate monitoring of the progression of the healing process without uncovering the wound. The innermost layer, comprised of Carbapol-PEO, is responsible for both the adhesion of the bandage to the wound and also neutralizing the high alkalinity of wounds, especially diabetic wounds. The first electrospun nanofiber layer comprises Carbapol

PEO either alone or with antibiotic loaded hyalorosomes. The electrospinning parameters for this layer were adjusted to be 0.5 - 1 ml/hr feed rate, 12-15 KeV applied voltage, 15 cm distance between the spinneret tip and the collector and 35 - 50 % humidity.

Any PLLA layer containing either indicator or drug(s) was electrospun using the following parameters: 4-5 ml/hr feed rate, 15-17 KeV applied voltage, 15 cm distance between the spinneret tip and the collector, and 50 - 75 % applied voltage. The humidity was increased during electrospinning of PLLA nanofibers to help increase the porosity on the surface of the PLLA nanofibers produced.

Different final bandages were produced to test the effect of each drug individually and in combination with others. Both the outermost and innermost layers are the same in all of them. The middle layer or layers all comprised PLLA but comprise different drugs or different drug combinations. The bandages produced are summarised in the table below:

Example	No. of middle layers	Drug(s) present
1	1	Phenytoin
2	1	NO releasing nanoparticles
3	1	Sildenafil
4	1	Simvastatin
5	2	Phenytoin
NO releasing nanoparticles
6	2	Phenytoin
Sildenafil
7	2	Phenytoin
Simvastatin
8	3	Phenytoin
Sildenafil
Simvastatin

Characterization of the Wound Bandage

Morphological Investigation of the Different Bandage Lavers

Scanning electron microscopy (SEM) was used to investigate the different PLLA nanofibers and Carbapol-PEO nanofibers produced by electrospinning. In particular SEM was used to determine which nanofibers were the most uniform and which had the highest porosity, both within the nanofiber matrices and on the surface of the nanofibers per se. SEM was also used to determine the optimum ratio between Carbapol and PEO that could be electrospun free of beaded regions.

Investigating the sensitivity of the PLLA-Indicator Laver

The sensitivity of the PLLA-indicator layer comprising methylene blue towards a change in the humidity percentage was investigated. Samples of the bandage were placed in different humidity environments using saturated solutions of different salts. Then the maximum absorbance of the indicator layer was detected using a UV-Vis spectrophotometer at different time points (0 min, 0 min, 30 min, 50 min, 80 min and 120 min). The salts forming the saturated solutions were lithium chloride (10% humidity), potassium acetate (20% humidity), magnesium chloride (30% humidity), magnesium nitrate (50% humidity), sodium chloride (75% humidity) and potassium sulphate (100% humidity).

Physicochemical Characterization

Physicochemical characterization was carried out to study the properties of the wound bandage that are related to the parameters required for fabrication of scaffolds generally and wound bandages specifically. These physicochemical tests are: biodegedability, swellability, water vapor permeability and porosity.

• Biodegradability

Biodegradability is an important parameter that should be investigated during scaffold fabrication to ensure the safety of the material and its durability. The biomaterial should be degraded easily and safely. In addition, it should be durable to some extent to tolerate the period of treatment without being torn out or destroyed before the end of treatment.

Biodegradability was tested by placing a known amount of the bandage material in a buffer of pH 7.4 for 30 days in a shaking incubator at room temperature. At certain time points, the bandage material was removed, dried, and weighed again before being placed again in the buffer medium. The weight remaining percentage was calculated using equation (1) then plotted against the different time points, where Wf is the final weight at each time point while Wi is the initial weight at the beginning of the experiment.

Reminaing Weight %= (Wf/Wi) *100 (1)

Swellability

Swellability is another parameter that is important to investigate the hydrophilicity of the biomaterial used. As the swelling ability of a material increases, its ability to release any drugs incorporated therein in a well-controlled and sustained manner increases.

The bandage material of a known weight was placed in a buffer medium of pH 7.4 and left in a shaking incubator at room temperature. The bandage material was removed, excess buffer was removed using a filter paper, and the bandage weighed again. This step was repeated until no increase in the material weight could be detected. Then the swellability percentage was calculated using equation (2), where Wd is the initial weight of the dry sample, while Ws is the weight of material after swelling at each time point. Swelling percentage was then plotted against different time points.

Swelling %= ((Ws-Wd)ZWd) x 100 (2) • Water Vapor Permeability

Water vapor permeability testing was carried out to investigate the breathability of the bandage material to ensure its capability to pass air to wound sites. Also, a high porosity stimulates the proliferation and differentiation of the damaged or lost cells.

Water vapor permeability was tested using ASTM E96 desiccant method. A known amount of desiccant (silica gel) was placed inside an Erlenmeyer flask whose top was closed by a piece of the bandage material. Then it was placed in a 75% humidity saturated environment for 30 days. The Water vapor permeation (WVP) through the bandage was then calculated using Equation (3) where Δ W is the change in weight due to water vapor permeation, A is the surface area and At is the change in time.

WVP = ((Δ W )/A) X At (3) • Porosity

The presence of high porosity within a scaffold is a mandatory condition to ensure a high rate of cell proliferation and tissue regeneration.

The porosity of the bandages was measured using a pycnometer (Ultrapyc 1200 e, Quantachrome instruments, USA). A piece of the bandage of known dimensions was used, allowing both of the porosity per volume and porosity percentage of the bandage to be calculated using Equations (4) and (5), respectively, where, VF is the volume of the bandage estimated by the pycnometer using helium gas.

Porosity per Volume=1- VF/(Total Volume (Length X Width X Thickness) (4) Porosity % =Porosity per Volume X 100 (5)

In Vitro Drug Release Experiments

Drug release profile experiments were performed for each drug used individually and in combination with others according to each bandage fabricated. All experiments were conducted using a buffer of pH 7.4 to simulate the expected pH of wounds after being neutralized with the carbapol layer. The amount of the drug released was estimated using UV-Vis spectrophotometer at different time points. The experiments were run for 2 - 3 weeks according to the drug used then the cumulative release percentage was calculated and plotted against the different time points.

In Vitro Cell Experiments

Cell Viability (Cytotoxicity)

The cytotoxicity of the bandages fabricated was estimated through the MTT (3(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide) assay. The experiment was conducted using human dermal fibroblasts for 24 hr. Then the experiment was terminated, before adding the coloring reagent and estimating the optical density of the viable cells. The percentage of the viable cells was then calculated using equation (6).

Cell Viability % =((Mean Optical Density)/(Control Optical Density)) *100 (6)

Cell Proliferation and Imaging

Human dermal fibroblasts were used to test the cell proliferation ability on the surface of the tested bandages. The cells were suspended in their growth medium then seeded in well pates for 48 hr. Then, the cells were fixed on the surface of the bandages and investigated using both an optical microscope and scanning electron microscope. These tests are used to confirm the capability of the bandages to be used as scaffolds fortissue regeneration.

In Vivo Animal Model

Laboratory animals

Animal experiments were performed according to institutional ethical guidelines. Approval of the wound healing animal model study was obtained from the Animal

Research Committee of Misr University for Science and Technology. A total of 180 male albino rats with an average weight of 150 ± 20 g were used for the experiment. The animals were kept in a well-ventilated house with free access to food and drinking water during the entire experimental period.

Induction of diabetes

Alloxan monohydrate of 150 mg/kg bodyweight was administered intraperitoneally to induce the diabetes. The fasting blood glucose levels of the rats were previously determined after 12 hr of fasting. After 48 hr of induction, the tail arteries of these animals were punctured to collect the blood and used to determine their blood glucose levels using Accu-check active glucometer. Rats with fasting blood glucose levels between 250 and 400 mg/dl were considered diabetic.

Animal grouping

The animals were randomly grouped as follows with eighteen albino rats in each group:

• Group I: No drug in nanofibrous layer.

• Group II: Phenytoin-loaded nanofibrous layer.

• Group III: Sildenafil-loaded nanofibrous layer • Group IV: Nitric oxide eluting nanoparticles-loaded nanofibrous layer.

• Group V: Simvastatin-loaded nanofibrous layer.

• Group VI: Phenytoin-loaded nanofibrous layer + Sildenafil-loaded nanofibrous layer.

• Group VII: Phenytoin-loaded nanofibrous layer + Nitric oxide eluting nanoparticlesloaded nanofibrous layer.

• Group VIII: Phenytoin-loaded nanofibrous layer + Simvastatin-loaded nanofibrous layer.

• Group IX: Phenytoin-loaded nanofibrous layer + Sildenafil-loaded nanofibrous layer + Simvastatin-loaded nanofibrous layer + Ciprofloxacin-loaded hyalurosomes nanofibrous layer.

• Group X: Commercial product (Phenytoin based spray)

All systems were multilayered where in the innermost, first layer was a Carbopol based nanofiber layer and the outermost, external layer was color sensor nanofiber layer.

Histopathological examination:

Skin specimens were fixed in 10% formol saline, then trimmed off, washed and dehydrated in ascending grades of alcohol. The dehydrated specimens were then cleared in xylene, embedded in paraffin blocks and sectioned at 4-6 pm thick. The obtained tissue sections were deparaffinized using xylol and stained using hematoxylin and eosin (H&E) for histopathological examination through the electric light microscope.

A semi-quantitative method was used to evaluate the following histological processes and structures: reepithelization, PMNL (polymorphonuclear leucocytes), fibroblasts, new vessels, and new collagen. Sections were evaluated according to the scale: 0, 1,2, 3, 4, explained in the table below.

Scale	Epithelization	PMNL	Fibroblasts	New vessels	Collagen
0	thickness of cut edges	absent	absent	absent	absent
1	migration of cells (< 50%)	mild ST	mild ST	mild-SCT	minimal-GT
2	migration of cells (> 50%)	mild DL/GT	mild-GT	mild-GT	mild-GT
3	bridging the excision	moderate DL/GT	moderate-GT	moderate-GT	moderate-GT
4	keratinization	marked DL/GT	marked-GT	marked-GT	marked-GT

Table 1: ST - surrounding tissue, i.e. tissue out of GT; DL - demarcation line; SCT subcutaneous tissue; GT - granulation tissue)

Results and Discussion

Morphological Examination of the Different Fabricated Nanofibers Layers • PLLA-Based Layer

SEM was usesd to assess the porosity of the PLLA-based nanofibers. Figure 3 shows SEM images of some of the nanofibers produced. Figure 3(a) shows PPLA nanofibers spun from DCM solution. Figure 3b shows nanofibers spun from DCM:DMF (1:1) solution. Figures 3c and 3d shows higher magnification images of nanofibers spun from DCM to show their highly porous surface.

It was found DCM was the best solvent for production of PLLA-based nanofibers by electrospinning. It led to the production of nanofibers with high porosity in the nanofiber matrix as well as on the nanofiber surface per se.

Carbapol-Based Layer

Solutions with different ratios of carbapol and PEO were fabricated using electrospinning. SEM was used to assess the nanofibers produced. Figure 4a shows pure carbapol nanofibers, Figure 4b) shows pure PEO nanofibers, Figure 4c) shows nanofibers with carbapol: PEO in a ratio of 75:25, Figure 4d) shows nanofibers with carbapol: PEO in a ratio of 50:50 and Figure 4e) shows nanofibers with carbapol: PEO in a ratio of 25:75. It was found that the ratio of 50:50 enabled the carbapol and PEO to be electrospun, without producing beads.

Sensitivity of PLLA-lndicator Laver to Different Humidity Ratios

The sensitivity of the PLLA layer containing methylene blue to moisture was determined by placing the bandage in environments with a different humidity percentile (10, 30, 50, 75 & 100%). The maximum absorbance of these bandages was then detected at different time intervals (minutes) using a UV spectrophotometer. As shown in Table 2 and in Figure 5a it was found that the layer is highly sensitive to the different humidity environment. This indicates that this layer may be used to indicate the progress of the healing process by changing its color with the change in the humidity during the healing process. This is very beneficial in monitoring the wound healing process underneath the bandage without removing it.

	Time 0 min	Time 10 min	Time min	30	Time 50 min	Time 80 min	Time 120 min
Moisture %	Absorbance
10	555±0	561±4	564+1	571±0	575±1	573±1
30	555±0	576±5	599+2	595±5	600±0	595±1
50	555±0	576±1	598+2	596±5	601±l	601±0
75	555±0	590±ll	601+1	603±l	602±l	602±l
100	555±0	596±0	607+1	611±0	611±0	611±0

Table 2

The bandage was also monitored visually for color change upon exposure to different humidity environments at different time intervals. As seen in Figure 5b it was facile to see the change in the color as a response to the change in humidity.

Biodegradability

The biodegradability of the bandage was monitored for 30 days and the results are shown in Figure 6. It is clear that both the PLLA-based layers and the carbapolPEO layer are biodegradable. Despite being biodegradable, the bandage was durable as it lost less than 20% of its weight during the whole month.

Swellability

The swellability of the bandage was examined to ensure its ability to release the drug(s) incorporated therein at appropriate times. The results are shown in Figure 7. Figure 7 shows that the different PLLA-based layers all reach their swelling maximum after only 2-3 hours. This is an advantage as it indicates the bandage will readily be able to start releasing the incorporated drug(s) in a short period of time after application of the bandage.

Water Vapor Permeability & Porosity

Water vapor permeability and porosity experiments proved that the bandage layers possess pores in their structure that exceed 95% of their volume. This confirms the ability of the bandage to permit the passage of nutrients and oxygen through the bandage thereby allowing cell proliferation and regeneration.

In Vitro Drug Release Experiment

The results showed that the bandage acted as a successful controlled and sustained release carrier for the model drugs incorporated into its structure. This can be observed in Figure 8. It can also be seen that lecithin increases the rate of the drug(s) release. This can be used to manipulate the bandage according to the wound being treated.

In Vitro Cell Experiments

Cell Viability

It was found that all of the materials used in the bandage are high biocompatible (Figure 9). This was confirmed through performing the MTT assay which showed that the bandage not only preserved the cells but also stimulated its regeneration and proliferation.

Cell Proliferation and Imaging

Cell proliferation was examined through seeding cell suspensions in 24-well plates containing parts of the bandage. The cells were then fixed and imaged using SEM as illustrated in Figure 10. It was found that the cells were attached successfully on the surface of the bandage which confirms the ability of the bandage to act as a successful scaffold for skin regeneration.

In vivo animal study

The results are shown in Tables 3 below and in Figure 11 and are summarised below.

Table 3a: Effect of Phenytoin and sildenafil combination

G r 0 u P s	Histological scale of wound healing
£pithelization	PMNL	Fibroblasts	New vessels	Collagen
D
y
1	0	0	0	0	2	4	4	4	4	4	2	2	2	2	3	3	3	4	1	1	3	3	1	0	1	1	2	2	3	4
2	0	0	0	2	4	4	4	4	4	2	1	0	2	2	3	2	1	1	1	2	2	4	1	1	1	1	2	3	4	4
1 0	0	0	1	2	3	4	4	4	3	2	1	0	2	1	1	2	1	2	0	3	3	3	3	0	0	1	2	3	3	4
3	0	0	0	2	4	4	4	4	2	1	0	0	2	1	3	2	3	1	0	1	3	4	1	1	1	1	2	3	3	4
6	0	0	1	2	4	4	4	4	3	1	0	0	2	1	1	2	1	1	0	1	3	3	3	0	0	1	2	3	3	4

Table 3b: Effect of Phenytoin and Nitric oxide eluting nanoparticles combination

G r 0 u P s	Histological scale of wound healing
Epithelization	PMNL	Fibroblasts	New vessels	Collagen
D a y			Γ	r-	r-	is			Γ-	r-	r-	is			Γ-	r-	r-	is			Γ-	r-	r-	is			Γ-	i—	i—	is
1	0	0	0	0	2	4	4	4	4	4	2	2	2	2	3	3	3	4	1	1	3	3	1	0	1	1	2	2	3	4
2	0	0	0	2	4	4	4	4	4	2	1	0	2	2	3	2	1	1	1	2	2	4	1	1	1	1	2	3	4	4
1 0	0	0	1	2	3	4	4	4	3	2	1	0	2	1	1	2	1	2	0	3	3	3	3	0	0	1	2	3	3	4
4	0	0	1	2	3	4	4	4	3	1	0	0	2	1	1	2	1	1	0	1	1	3	1	0	0	1	2	3	3	4

7

0

1

2

4

3

2

1

0

2

1

2

1

2

0

3

2

0

1

2

3

4

Table 3c: Effect of Phenytoin and Simvastatin combination

G r 0 u P s	Histological scale of wound healing
Epithelization	PMNL	Fibroblasts	New vessels	Collagen
D
a y			Γ	o		tN			Γ-	o		tN			Γ-	o		tN			Γ-	o		tN			Γ-	o		tN
1	0	0	0	0	2	4	4	4	4	4	2	2	2	2	3	3	3	4	1	1	3	3	1	0	1	1	2	2	3	4
2	0	0	0	2	4	4	4	4	4	2	1	0	2	2	3	2	1	1	1	2	2	4	1	1	1	1	2	3	4	4
1 0	0	0	1	2	3	4	4	4	3	2	1	0	2	1	1	2	1	2	0	3	3	3	3	0	0	1	2	3	3	4
5	0	0	1	2	4	4	4	4	3	1	0	0	2	1	1	2	1	1	0	1	3	3	0	0	0	1	2	3	3	4
8	0	0	1	3	3	4	4	4	3	2	1	0	2	1	1	2	1	2	0	3	3	3	2	2	0	1	2	3	3	4

Table 3d: Effect of Phenytoin, Sildenafil, Simvastatin and Ciprofloxacin combination

G r 0 u P s	Histological scale of wound healing
Epithelization	PMNL	Fibroblasts	New vessels	Collagen
D a y			I	o		tN			Γ-	o					Γ-	o		tN			Γ-	o					Γ-	o		C4
1	0	0	0	0	2	4	4	4	4	4	2	2	2	2	3	3	3	4	1	1	3	3	1	0	1	1	2	2	3	4
2	0	0	0	2	4	4	4	4	4	2	1	0	2	2	3	2	1	1	1	2	2	4	1	1	1	1	2	3	4	4
1 0	0	0	1	2	3	4	4	4	3	2	1	0	2	1	1	2	1	2	0	3	3	3	3	0	0	1	2	3	3	4
9	0	0	1	3	3	4	4	4	3	2	1	0	2	1	1	2	1	2	0	3	3	3	3	3	0	1	2	3	3	4

days of wound induction:

The animals of all treated groups showed thickening of epidermis at its cut edges. The dermis near the excision was rich on inflammatory cells (PMNL). The number of fibroblasts absence or mild increased in the dermis near the wounded area. The dermal layer displayed the beginning of neo-angiogenesis in most of examined groups. The presence of new collagen was not recorded.

days of wound induction:

All examined groups revealed fibrin net rich on inflammatory cells mainly neutrophils, macrophages and lymphocytes. The regeneration of the epidermis was completely inhibited. Mild proliferation and migration of fibroblasts and mild new collagen were observed.

days of wound induction:

By 7 days after wound induction the gap was filled with necrotic tissues and inflammatory cells, mainly neutrophils in group (1-2-3). The epidermis regeneration was significantly inhibited as well as delayed proliferation and migration of fibroblasts. The general picture of healing was delayed due to a slightly inhibited both fibroblast and endothelial cells proliferation which accompanied with Lowering of new vessels number.

On other side groups (4-5-6-7-8-9-10) showed migration of cells (< 50%) and lightly infiltrated with PMNL, thus the inflammatory phase was almost finished. At the bottom of wounds newly created granulation tissue was observed. The granulation tissue consisted of fibroblasts, endothelial cells, and newly synthesized non-organized collagen.

days of wound induction:

By 10 days after wound induction, bridging the excision by epidermal basal layer were seen in groups 2 and 9. Mature granulation tissues with very mild polymorphonuclear cells infiltration. In the other groups, migration of basal epidermal cells, with different degree and moderate polymorphonuclear cells infiltration, was observed.

days of wound induction:

By 14 days after wound induction, the regeneration of the epidermis was finished in all groups except group (1). the differentiation process of keratinocytes was confirmed by the normal process of the keratinization in group (2-3-5-6). During this time the tissue macrophages predominant from the inflammatory cell’s population. The number of fibroblasts and endothelial cells in the granulation tissue decreased and increase of collagen fibers.

days of wound induction:

All animal group specimens showed complete organization of granulation tissue and scar was created. The inflammatory phase, angiogenesis and fibroblast cells proliferation were finished.

Claims

1. A wound healing system comprising:

(i) a mucoadhesive layer comprising a nanofiber matrix;

2. A system as claimed in claim 1, wherein said mucoadhesive layer comprises nanofibers having an average diameter of 50 to 4000 nm.

3. A system as claimed in claim 1 or 2, wherein said mucoadhesive layer comprises a nanofiber matrix comprising a mucoadhesive polymer.

4. A system as claimed in claim 3, wherein said mucoadhesive polymer comprises at least one monomer having -COOH side groups.

5. A system as claimed in claim 3 or 4, wherein said mucoadhesive polymer is selected from carbomers (polyacrylic acid), polyethylene oxide, polyacrylates, polyvinyl alcohol, polyalkylene glycol, polycarbophil, polyoxyethlene-polyoxypropylene block copolymers, cellulose derivatives, natural polysaccharides, chitosan, gelatin, hyaluronic acid, alginitic acid and mixtures thereof.

6. A system as claimed in claim 5, wherein said mucoadhesive polymer is a mixture of carbomer and polyethylene oxide.

7. A system as claimed in any of claims 1 to 6, wherein said mucoadhesive layer comprises a nanofiber matrix comprising an antimicrobial agent.

8. A system as claimed in claim 7, wherein said antimicrobial agent is selected from moxifloxacin, natamycin, azythromycin, mupirocin, erythromycin, ciprofloxacin, netilmycin, besifloxacin, gatifloxacin, gentamycin sulfate, levofloxacin, ofloxacin, sulfacetamide sodium, tobramycin, bacitracin zinc, Polymyxin B sulfate, neomycin, and neomycin sulfate, acyclovir, valacyclovir, famciclovir, itraconazole, posaconazole, voriconazole and silver sulfadiazine.

9. A system as claimed in any one of claims 1 to 8, wherein said mucoadhesive layer has a thickness of 250-2000 μητ

10. A system as claimed in any preceding claim, wherein said indicator layer comprises nanofibers having an average diameter of 50 to 4000 nm.

11. A system as claimed in any preceding claim, wherein said indicator layer comprises a nanofiber matrix comprising a polymer selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid, poly-D-lactic acid, poly-L-lactic acid, PLGAdimethacrylate, fluorescent PLGA polymers, a poly(meth)acrylate, poly(methyl)methacrylate, poly(hydroxymethyl)methacrylate, a polyanhydride, a polyorthoester, a polyetherester, a polycaprolactone, a polysaccharide, a polyester, a polydioxanone, a polygluconate, an ethyl cellulose, cellulose derivatives, chitosan derivatives or mixtures or combinations thereof.

12. A system as claimed in claim 11, wherein said polymer is selected from polylactic acid, poly-D-lactic acid, poly-L-lactic acid or mixtures thereof.

13. A system as claimed in any preceding claim, wherein said moisture indicator is selected from methylene blue, acyl auramines, acylleucophenothiazines, alpha- and beta-unsaturated aryl ketones, azaphthalides, basic mono azo dyes, 10-benzoylN,N,N',N'-tetraethyl-3,7-diamino-10H-phenoxazine, chromogenic azaphthalide compounds, diaryl phthalides, diphenylmethanes, dithio-oxamide, di[bis(indolyl)ethylenyl]tetrahalophthalides, fluoran derivatives (3-dialkylamino-7dialkylamylfluoran), 3-(indol-3-yl)-3-(4-substituted aminophenyl)phthalides, bis(indolyl)ethylenes, indolyl red, leucoauramines, 3-methyl-2,2-spirobi(benzo-[f]chromene), phenoxazine, phthalides including crystal violet lactone, malachite green lactone, phthalide red, phthalide violet, phthalans, benzoindolinospiropyrans, rhodamine beta lactams, spiropyrans, triphenylmethanes including gentian violet and malachite green.

14. A system as claimed in any one of claims 1 to 13, wherein said indicator layer has a thickness of 50-4000 μητ

15. A system as claimed in any preceding claim, wherein said first drug-containing layer comprises nanofibers having an average diameter of 50 to 4000 nm.

16. A system as claimed in any preceding claim, wherein said first drug-containing layer comprises a nanofiber matrix comprising a polymer selected from poly(lactic-coglycolic acid) (PLGA), polylactic acid, poly-D-lactic acid, poly-L-lactic acid, PLGAdimethacrylate, fluorescent PLGA polymers, a poly(meth)acrylate, poly(methyl)methacrylate, poly(hydroxymethyl)methacrylate, a polyanhydride, a polyorthoester, a polyetherester, a polycaprolactone, a polysaccharide, a polyester, a polydioxanone, a polygluconate, an ethyl cellulose, cellulose derivatives, chitosan derivatives or mixtures or combinations thereof.

17. A system as claimed in claim 16, wherein said first drug-containing layer comprises a nanofiber matrix comprising a polymer selected from polylactic acid, polyD-lactic acid, poly-L-lactic acid or mixtures thereof.

18. A system as claimed in any preceding claim, wherein said first drug-containing layer comprises a nanofiber matrix comprising a drug selected from an antiflammatory, an angiogenesis stimulator or a proliferation promoter.

19. A system as claimed in any one of claims 1 to 18, wherein said first drugcontaining layer has a thickness of 250-2000 μητ

20. A system as claimed in any preceding claim, further comprising a second drugcontaining layer comprising a nanofiber matrix.

21. A system as claimed in claim 20, wherein said second drug-containing layer comprises a nanofiber matrix comprising a drug selected from an antiflammatory, an angiogenesis stimulator or a proliferation promoter.

22. A system as claimed in claim 20 or 21, wherein said second drug-containing layer has a thickness of 250-2000 μητ

23. A system as claimed in any preceding claim, further comprising a third drugcontaining layer comprising a nanofiber matrix.

24. A system as claimed in claim 23, wherein said third drug-containing layer comprises a nanofiber matrix comprising a drug selected from an antiflammatory, an angiogenesis stimulator or a proliferation promoter.

25. A system as claimed in claim 23 or 24, wherein said third drug-containing layer has a thickness of 250-2000 μητ

26. A system as claimed in any one of claims 1 to 25, wherein said first drugcontaining layer comprises a nanofiber matrix comprising a proliferation promoter.

27. A system as claimed in any one of claims 20 to 26, wherein said second drugcontaining layer comprises a nanofiber matrix comprising an angiogenesis stimulator.

28. A system as claimed in any one of claims 23 to 27, wherein said third drugcontaining layer comprises a nanofiber matrix comprising an anti-inflammatory drug.

29. A system as claimed in any one of claims 20 to 28 comprising a first and a second drug-containing layer, wherein the order of the layers in said system is:

(a) mucoadhesive layer;

(b) first drug-containing layer;

(c) second drug-containing layer; and (d) indicator layer.

30. A system as claimed in any one of claims 23 to 29 comprising first, second and third drug-containing layers, wherein the order of the layers in said system is:

(a) mucoadhesive layer;

(b) first drug-containing layer;

(c) second drug-containing layer;

(d) third drug-containing layer; and (d) indicator layer.

31. A system as claimed in any one of claims 1 to 30, which is a wound dressing.

32. A system as claimed in any one of claims 1 to 31, wherein said system has a thickness of 0.5 to 10 mm.

33. A system as claimed in any one of claims 1 to 32, further comprising a release liner.

34. A system as claimed in any one of claims 1 to 33, further comprising a backing layer.

35. A method of making a wound healing system as claimed in any one of claims 1 to 34, comprising:

36. A wound healing system as claimed in any one of claims 1 to 34 for use in the treatment of wounds.

37. A system as claimed in claim 36, wherein said wound is selected from a burn, an abrasion, a laceration, a puncture, an avulsion, an incision, a graft, a lesion caused by an infectious agent, a chronic venous ulcer, a diabetic ulcer, a compression or decubitus ulcer and a mucosal ulcer.

38. Use of at least a first drug in the manufacture of a wound healing system as claimed in any one of claims 1 to 35 for the treatment of wounds.

39. A method of treating a wound in a patient in need thereof, comprising:

(i) placing a wound healing system as claimed in any one of claims 1 to 34 onto a wound on said patient; and (ii) maintaining said system on said wound for a therapeutically effective period of time.