GB2570113A - Ocular drug delivery system - Google Patents

Abstract

An ocular drug delivery system comprising a nanofiber matrix, wherein said nanofiber matrix comprises drug-containing nanoparticles. Preferably, the nanoparticles are impregnated in the nanofiber matrix. The nanoparticles may comprise a biodegradable, hydrophobic polymer, most preferably poly(lactic-co-glycolic acid) (PLGA); and/or a biodegradable, amphiphilic polymer selected from various polyethylene glycol block co-polymers. Preferably, the drug is selected from an anti-microbial, anti-inflammatory, anti-glaucoma, analgesic, an anaesthetic or combinations thereof, an exemplified drug is azithromycin. The nanofiber may comprise a mucoadhesive polymer, preferably polyvinylpyrrolidone. The drug delivery system may be an ocular insert, preferably a patch. Preferably, the system of the invention provides controlled-release of drug over a period of at least 3 days. A method of making said ocular drug delivery system is also disclosed; comprising preparing nanofibers impregnated with drug-containing nanoparticles, preferably by electrospinning; then forming the resulting nanofibers into said ocular drug delivery system. Also claimed are an ocular drug delivery system for use in the treatment of ocular diseases and a method of treating an ocular disease in a patient, comprising placing said ocular drug delivery system into the eye of said patient and maintaining said system in said eye for a therapeutically effective period of time.

Description

Ocular Drug Delivery System

INTRODUCTION

The present invention relates to an ocular drug delivery system comprising a nanofiber matrix, wherein the nanofiber matrix comprises drug-containing nanoparticles. The invention also relates to a method of making the ocular drug delivery system and to medical uses of the ocular drug delivery system for the treatment of ocular diseases such as microbial infections and glaucoma.

BACKGROUND

The majority of ophthalmic drugs are administered in the form of eye drops. Eye drops, however, have limited therapeutic effect due to the anatomy, physiology, and biochemistry of the eye that act as protective mechanisms against foreign bodies. Traditional eye drops are, for example, rapidly eliminated due to high tear turnover and have a precorneal residence time of about 1-3 min that allows only ~3% of the applied drug dose to penetrate through the cornea to reach intraocular tissues.

Furthermore, many ophthalmic drugs are small, hydrophobic molecules which have poor solubility in water. For this reason, many liquid eye drops are drug suspensions. This exacerbates the bioavailability problem because, in this case, the drug must first solubilise in the eye and then be absorbed. The fast elimination rate means, however, that a very low absorption rate of drug is actually achieved.

Several studies have been published using different approaches to improve the bioavailability of ophthalmic drugs. These include the use of drug-containing ocular inserts which are placed in the eye. These offer an attractive alternative to eye-drops as they solve the difficult problem of limited pre-corneal residence time. Additionally the release of drug may be at a slow, constant rate, which, in turn, reduces the amount of systemic absorption. On the other hand, it has been found that a number of ocular inserts currently available have low patient compliance due to the fact that they are “felt” by the patients as an extraneous body in the eye. In the event that the insert moves around the eye it may also interfere with vision and make removal of the insert more difficult.

SUMMARY OF INVENTION

Viewed from a first aspect the present invention provides an ocular drug delivery system comprising a nanofiber matrix, wherein said nanofiber matrix comprises drug-containing nanoparticles.

Viewed from a further aspect the present invention provides a method of making an ocular drug delivery system as hereinbefore described comprising:

(ii) preparing nanofibers impregnated with drug-containing nanoparticles, preferably by electrospinning; and (iii) forming the resulting nanofibers into said ocular drug delivery system.

Viewed from a further aspect the present invention provides an ocular drug delivery system for use in the treatment of ocular diseases.

Viewed from a further aspect the present invention provides the use of a drug in the manufacture of an ocular drug delivery system as hereinbefore described for the treatment of ocular diseases.

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

(i) placing an ocular drug delivery system as hereinbefore described into the eye of said patient; and (ii) maintaining said system in said eye for a therapeutically effective period of time.

DEFINITIONS

As used herein the term “drug delivery system” refers to any system that is used as a medium or carrier for administering a pharmaceutical product to a patient.

As used herein the term “ocular drug delivery system” refers to a drug delivery system which administers a pharmaceutical product to a patient via an eye or any part thereof.

As used herein the terms “sustained release delivery system” and “controlled release delivery system” are used interchangeably to refer to a delivery system designed to provide a continuous release of a therapeutic agent over a period of time. Sustained and controlled release delivery systems advantageously provide a relatively consistent concentration of the therapeutic agent in vivo.

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

As used herein the term “nanoparticle” refers to a particle having an average diameter of less than 1000 nm.

As used herein the term “biodegradable” refers to a polymer(s) which will break down in vivo. The degradation process concurrently releases drug.

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

As used herein the term “ocular disease” refers to any disease or condition that affects any area of the eyeball, including the anterior and posterior segment of the eye.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an ocular drug delivery system comprising a nanofiber matrix, wherein the nanofiber matrix comprises drug-containing nanoparticles. The nanofiber matrix provides mucoadhesiveness that enables the delivery system to remain on the ocular surface for extended periods of time. Preferably the drug-containing nanoparticles are impregnated or encapsulated within the nanofiber matrix. Thus advantageously the nanofiber matrix provides a large surface area for the impregnation and/or encapsulation of the drug-containing nanoparticles. The nanoparticles and nanofibers together control the release rate of drug from the delivery system and sustained or controlled release delivery systems may be provided. Moreover the specific sustained or controlled drug release profile achieved may be tailored by modifying the composition of the nanoparticles.

In preferred delivery systems of the present invention the nanoparticles have an average diameter of 30-450 nm, more preferably 50 to 300 nm and still more preferably 60 to 100 nm. Preferably the average diameter of nanoparticles is the Z-average diameter. Preferably the average diameter of the nanoparticles is measured by dynamic light scattering, e.g. using a Zetasizer as described in the examples. In preferred delivery systems of the present invention the nanoparticles have a PDI of 0. ΙΟ.8, more preferably 0.15-0.6 and still more preferably 0.2-0.50. Preferably the nanoparticles have a relatively narrow particle size distribution. A narrow particle size distribution helps to ensure a consistent release rate of drug from the nanoparticles since the rate of a diffusion of the drug partially depends upon its path length.

The use of nanoparticles in the delivery system of the present invention provides a large surface area for the impregnation or encapsulation of drug. The nanoparticles therefore enable drug delivery systems of relatively small size to comprise doses of drug that are therapeutically effective for extended periods of time, particularly as the nanoparticles also control the release rate of the drug once it is administered.

In preferred delivery systems of the present invention, the nanoparticles comprise a hydrophobic polymer and preferably a biodegradable, hydrophobic polymer. Suitable polymers are commercially available. The presence of a hydrophobic polymer in the delivery system enables the release rate of drug from the nanoparticles to be controlled, specifically retarded. This is beneficial as it enables delivery systems that release drug over long periods of time, e.g. weeks or even months, to be prepared.

Preferably the hydrophobic polymer is selected from poly(lactic-co-glycolic acid), polylactic acid, poly-D-lactic acid, poly-L-lactic acid, PLGA-dimethacrylate, fluorescent PLGA polymers, a poly(meth)acrylate, 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 hydrophobic polymer is selected from poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly-D-lactic acid (PDLA), poly-L-lactic acid (PLLA) and PLGA-dimethacrylate. Still more preferably the hydrophobic polymer is poly(lactic-co-glycolic acid) (PLGA).

In preferred PLGA, the weight ratio of polylactic acid and polyglycolic acid monomers is 90:10 to 10:90, more preferably 75:25 to 25:75 and yet more preferably 65:35 to 35:65. In preferred PLGA the terminal groups are carboxylic, amino or a mixture thereof.

Preferably the hydrophobic polymer has a molecular weight (Mw) of 25,000 to 140,000 Dalton, more preferably 30,000 to 120,000 Dalton and still more preferably 40,000 to 100,000 Dalton.

In preferred delivery systems of the present invention, the nanoparticles comprise an amphiphilic or hydrophilic polymer and more preferably a biodegradable, amphiphilic or hydrophilic polymer. Preferably the nanoparticles comprise a amphiphilic polymer and particularly preferably a self-assembling amphiphilic polymer. The presence of an amphiphilic and/or hydrophilic polymer in the delivery system enables the release rate of drug from the nanoparticles to be tailored or modified. Compared to the hydrophobic polymer, this polymer increases the release rate of drug from the nanoparticles. Thus by controlling the precise nature of each of the hydrophobic and amphiphilic polymers as well as their ratio, the rate of release of drug from the nanoparticles may be controlled.

Preferably the amphiphilic polymer is selected from block copolymers of ethylene glycol-propylene glycol-ethylene glycol (PEG-PPG-PEG), polylactic acidethylene glycol-polylactic acid (PLA-PEG-PLA), poly(lactic-co-glycolic acid)-ethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) and polyglycolic acid-ethylene glycol-polyglycolic acid (PGA-PEG-PGA). Suitable polymers are commercially available. Representative examples of suitable commercially available polymers are those sold under the tradename, Pluronics (e.g. Pluronic® F-127) and Synperonics.

Particularly preferably the amphiphilic polymer is selected from block copolymers of ethylene glycol-propylene glycol-ethylene glycol (PEG-PPG-PEG). Such polymers are sometimes referred to as poloxamers. Preferred block copolymers of ethylene glycol-propylene glycol-ethylene glycol comprise ethylene glycol and propylene glycol monomers in a weight ratio of 9:1 to 1:1, more preferably 6:1 to 3:1 and yet more preferably 5:1 to 3:1.

Preferably the amphiphilic polymer has a molecular weight (Mw) of 20,000 to 80,000, more preferably 30,000 to 70,000 and still more preferably 40,000 to 60,000.

In particularly preferred delivery systems of the present invention, the nanoparticles comprise a mixture of at least one hydrophobic polymer and at least one amphiphilic polymer. More preferably the nanoparticles comprise a mixture of poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), poly-D-lactic acid (PDLA), poly-L-lactic acid (PLLA) and PLGA-dimethacrylate and a block copolymer of ethylene glycol-propylene glycol-ethylene glycol (PEG-PPG-PEG), polylactic acid-ethylene glycol-polylactic acid (PLA-PEG-PLA), poly(lactic-co-glycolic acid)-ethylene glycolpoly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) and polyglycolic acid-ethylene glycolpolyglycolic acid (PGA-PEG-PGA). Still more preferably the nanoparticles comprise a mixture of poly(lactic-co-glycolic acid) (PLGA) and a block copolymer of ethylene glycol-propylene glycol-ethylene glycol (PEG-PPG-PEG) .

In further particularly preferred delivery systems of the present invention the weight ratio of hydrophobic polymer to amphiphilic polymer is 5:1 to 1:5, more preferably 3:1 to 1:3 and yet more preferably 2:1 to 1:2. Especially preferably the weight ratio of hydrophobic polymer to amphiphilic polymer is about 1:1.

Without wishing to be bound by theory, it is thought that the drug is impregnated within the hydrophobic polymer and that the drug is encapsulated within micelles formed by the amphiphilic polymer. When a mixture of hydrophobic and amphiphilic polymer is present it is thought that the amphiphilic polymer may also surround the hydrophobic drug-containing particles.

In preferred delivery systems of the present invention, the nanoparticles further comprise an ophthalmic drug. Preferably the ophthalmic drug 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, voraconazole, oxymetazoline hydrochloride, cetirizine hydrochloride, amfenac, nepafenac, lifitegrast, aspirin, ibuprofen, ketorolac, flurbiprofen, diclofenac, fluticasone propionate, fluticasone furoate, acetazolamide, echothiophate iodide ophthalmic, carbachol ophthalmic, apraclonidine, mitomycin, pilocarpine, nadolol, apraclonidine, methazolamide, brimonidine, timolol, bromfenac sesquihydrate, amfenac, nepafenac, aspirin, ibuprofen, ketorolac, tromethamine, diclofenac lidocaine and novocaine. More preferably the ophthalmic drug 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, voraconazole, oxymetazoline hydrochloride, cetirizine hydrochloride, amfenac, nepafenac, lifitegrast, aspirin, ibuprofen, ketorolac, flurbiprofen, diclofenac, fluticasone propionate, fluticasone furoate, acetazolamide, echothiophate iodide ophthalmic, carbachol ophthalmic, apraclonidine, mitomycin, pilocarpine, nadolol, apraclonidine, methazolamide, brimonidine and timolol. Particularly preferably the ophthalmic drug 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, voraconazole, acetazolamide, echothiophate iodide ophthalmic, carbachol ophthalmic, apraclonidine, mitomycin, pilocarpine, nadolol, apraclonidine, methazolamide, brimonidine and timolol.

In preferred delivery systems of the present invention, the nanoparticles further comprise a drug selected from an anti-microbial drug, an anti-inflammatory drug, an anti-glaucoma drug, an analgesic, an anaesthetic or combinations thereof. Particularly preferably the nanoparticles further comprise a drug selected from an anti-microbial drug, an anti-inflammatory drug, an anti-glaucoma drug, an ophthalmic drug or combinations thereof.

Preferred anti-microbial drugs 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, and voraconazole. Particularly preferred anti-microbial drugs are selected from levofloxacin, natamycin, ciprofloxacin, tobramycin and polymyxin.

Preferred anti-inflammatory drugs are selected from oxymetazoline hydrochloride, cetirizine hydrochloride, amfenac, nepafenac, lifitegrast, aspirin, ibuprofen, ketorolac, flurbiprofen, diclofenac, fluticasone propionate, and fluticasone furoate.

Preferred anti-glaucoma drugs are prostaglandins. Preferred anti-glaucoma drugs are selected from acetazolamide, echothiophate iodide ophthalmic, carbachol ophthalmic, apraclonidine, mitomycin, pilocarpine, nadolol, apraclonidine, methazolamide, brimonidine and timolol.

Preferred analgesics are selected from bromfenac sesquihydrate, amfenac, nepafenac, aspirin, ibuprofen, ketorolac, tromethamine and diclofenac.

Preferred anaesthetics are selected from lidocaine and novocaine.

In preferred delivery systems of the present invention the nanoparticles comprise 5-50 wt% of drug, more preferably 10 to 45 wt% of drug and still more preferably 12.5 to 40 wt% of drug, based on the total weight of the nanoparticles.

Optionally the nanoparticles further comprise surfactants, lubricants and/or wound healing components. Preferably, however, the nanoparticles consist of hydrophobic polymer, amphiphilic polymer and drug.

As described above, in the drug delivery system of the present invention the nanoparticles of the present invention are impregnated or encapsulated within a nanofiber matrix. 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 delivery systems of the present invention the nanofibers comprise a mucoadhesive polymer. This facilitates the retention of the delivery system in the eye at the site it is administered. Suitable biocompatible, mucoadhesive polymers are commercially available.

Preferably the mucoadhesive polymer is selected from polyvinylpyrrolidone, copovidone, copolymers of substituted vinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, cellulose polymers, chitosan, chitosan derivatives, polyethylene glycols (PEG), polypropylene glycols (PPG) and natural gums. More preferably the mucoadhesive polymer is selected from polyvinylpyrrolidone, copovidone, copolymers of substituted vinyl pyrrolidone, chitosan derivatives, and polyvinyl alcohol. Yet more preferably the mucoadhesive polymer is polyvinvylpyrrolidone.

Preferably the mucoadhesive polymer has a molecular weight (Mw) of 15,000 to 900,000 Dalton, more preferably 100,000 to 700,000 Dalton and still more preferably 250,000 to 480,000.

In preferred delivery systems of the present invention the nanofibers comprise 1-50 wt% of nanoparticles, more preferably 5 to 45 wt% of nanoparticles and still more preferably 10 to 40 wt% of nanoparticles, based on the total weight of the nanofiber matrix. In further preferred delivery systems of the present invention the nanofibers comprise 0.5-30 wt% of drug, more preferably 1 to 25 wt% of drug and still more preferably 2 to 20 wt% of drug, based on the total weight of the nanofiber matrix. In further preferred delivery systems of the present invention, the nanoparticles are homogeneously distributed within the nanofibers. Optionally the nanofibers further comprise surfactants, lubricants and/or wound healing components.

The ocular drug delivery system of the present invention is preferably in the form of an ocular insert and still more preferably in the form an electrospun ocular insert. Particularly preferably the ocular drug delivery system of the invention is a patch. Preferably the ocular insert is solely formed from the nanofiber matrix comprising drug-containing nanoparticles, i.e. the ocular insert does not comprise any additional layers or materials.

Preferably the cross section of the ocular insert is circular, square or rectangular. Preferably the insert, e.g. patch, is sized and shaped to readily fit into the eye, or a part thereof. Preferably the ocular insert has a thickness of 0.01-0.5 mm, more preferably 0.05-0.25 mm and still more preferably 0.1-0.2 mm.

Preferably the drug delivery system of the present invention has a porosity, e.g. as measured according to the method described in the examples, of at least 95 % and more preferably at least 98 %. This level of porosity ensures that the oxygen and lachrymal fluids can flow after placement of the delivery system into the eye.

Preferably the drug delivery 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 60 minutes, of at least 50 %wt and more preferably at least 60 %wt. Thus in preferred drug delivery systems of the present invention, less than 50 %wt and preferably less than 40 %wt of the drug delivery system remains after exposure to PBS for 60 minutes. This level of in vitro biodegradability indicates that the drug delivery system is well suited for in vivo use.

Preferably the drug delivery system of the present invention achieves its maximum swelling in PBS within 1 hour of being placed in the PBS, e.g. as determined according to the method in the examples. Preferably the ocular insert of the present invention achieves a swelling of at least 150 %wt, and more preferably at least 200 %wt in PBS within 1 hour of being placed in the PBS, e.g. as determined according to the method in the examples.

Preferably the drug delivery system of the present invention has a water vapour permeability of at least 250 mg.cm'².hr, and more preferably at least 300 mg.cm'².hr, after being placed in an eye for 60 minutes e.g. as determined by the method described in the examples herein.

An advantage of the drug delivery 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 both of the nanoparticles and the nanofibers. As a result, and despite the relatively small size of the drug delivery systems of the present invention, a delivery system (e.g. patch) preferably comprises an amount of drug that is required for a complete treatment regime. In other words, the delivery system provides a single dose treatment regimen.

A further advantage of the drug delivery systems of the present invention is that the system provides controlled-release of drug over a period of at least 3 days, more preferably at least 5 days and still more preferably at least 10 days. Preferably the system provides controlled-release of drug over a period of 3-15 days, more preferably 3 to 12 days and still more preferably 3-10 days. As mentioned above, an advantage of the delivery system of the present invention is that the drug release rate may be modified by selection of the polymers used to form the nanoparticles.

The present invention also relates to a method of making an ocular drug delivery system as hereinbefore described comprising:

(ii) preparing nanofibers impregnated with drug-containing nanoparticles, preferably by electrospinning; and (iii) forming the resulting nanofibers into the ocular drug delivery system.

Preferred methods of the invention further comprise the steps of:

(i) preparing drug-containing nanoparticles, preferably by nanoprecipitation. Thus particularly preferred methods of the invention comprise:

(i) preparing drug-containing nanoparticles, preferably by nanoprecipitation (ii) preparing nanofibers impregnated with drug-containing nanoparticles, preferably by electrospinning; and (iii) forming the resulting nanofibers into the ocular drug delivery system.

The drug-containing nanoparticles are preferably prepared by nanoprecipitation. Nanoprecipitation is a well known technique and the skilled person would readily understand what is involved. Preferably a solution of drug, hydrophobic polymer and amphiphilic polymer (e.g. as hereinbefore defined) in an organic solvent is prepared and then poured into an aqueous solution, preferably comprising surfactant or capping agent. The nanoparticles form spontaneously. Preferably the nanoparticles are recovered after complete evaporation of the organic solvent.

The nanofibers impregnated with drug-containing nanoparticles are preferably prepared by electospinning. Again this is a well known technique and the skilled person would readily understand what is involved. Preferably a solution of the mucoadhesive polymer and the drug-containing nanoparticles is prepared and then electrospun. 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 1-2.5 ml/hr;

Applied voltage: 10-50 kV, preferably 15-35 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: 25-40 °C, preferably 30-35 °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 drug delivery system. If necessary, the matrix produced by electrospinning may be cut to the desired shape and/or size. The drug delivery system of the present invention is preferably appropriately sized to fit into the conjunctival culde-sac of the eye.

The ocular drug delivery systems of the present invention are for use in the treatment of ocular diseases. Alternatively the present invention provides the use of a drug (e.g. as hereinbefore described) in the manufacture of an ocular drug delivery system as hereinbefore described for the treatment of ocular diseases. In a further alternative the present invention provides a method of treating an ocular disease in a patient in need thereof, comprising:

(i) placing an ocular drug delivery system as hereinbefore described into the eye of the patient; and (ii) maintaining said system in said eye for a therapeutically effective period of time.

Representative examples of ocular diseases that may be treated include glaucoma, blepharitis, corneal tear/injury, dry eye, aphakia, pseudophakia, astigmatism, blepharospasm, cataract, conjunctival diseases, conjunctivitis, allergic conjunctivitis, corneal diseases, corneal keratitis, corneal ulcer, dry eye syndromes, eyelid diseases, lacrimal apparatus diseases, lacrimal duct obstruction, myopia, presbyopia, pupil disorders, refractive disorders, strabismus, acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, infections such as bacteria, fungal or viral infections, macular degeneration (e.g., acute macular degeneration, non-exudative age related macular degeneration and exudative age related macular degeneration), edema (e.g., macular edema, cystoid macular edema and diabetic macular edema), multifocal choroiditis, ocular trauma which affects a posterior ocular site or location, ocular tumors, retinal disorders such as central retinal vein occlusion, diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease, sympathetic ophthalmia, Vogt Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused by or influenced by an ocular laser treatment, posterior ocular conditions caused by or influenced by a photodynamic therapy, post-surgical inflammation (cataract, glaucoma, corneal transplant), photocoagulation, radiation retinopathy, epiretinal membrane disorders, branch retinal vein occlusion, anterior ischemic optic neuropathy, nonretinopathy diabetic retinal dysfunction, and retinitis pigmentosa.

DETAILED DESCRIPTION OF THE FIGURES

Figure 1(a) shows a SEM image of AZT-loaded nanoparticles;

Figure 1(b) shows a 3D model of z-stacks of CLSM images of FDA-loaded nanoparticles

Figure 1(c) shows a SEM image of unloaded PVP nanofibers;

Figure 1(d) shows a histogram of unloaded PVP nanofiber diameters;

Figure 1(e) shows a 3D simulation of the upper view of unloaded PVP nanofibers;

Figure 1(f) shows a 3D simulation of the lower view of unloaded PVP nanofibers;

Figure 1(g) shows a SEM image of loaded PVP nanofibers;

Figure 1(h) shows a histogram of loaded PVP nanofiber diameters;

Figure 1(i) shows a 3D simulation of the upper view of loaded PVP nanofibers;

Figure 1(j) shows a 3D simulation of the lower view of loaded PVP nanofibers;

Figure 2 shows the FTIR Spectra of AZT, PL NPs, PLGA NPs, AZT-PL NPs, AZT-PLGA NPs and AZT-PL/PLGA NPs;

Figure 3a shows DSC thermograms of AZT, unloaded NPs, loaded NPs, raw PVP, unloaded NFs and loaded NFs;

Figure 3b shows TGA thermograms of AZT, unloaded NPs, loaded NPs, raw PVP, unloaded NFs and loaded NFs;

Figure 4 shows the XRD diffraction pattern of AZT, PL, PLGA, and AZTPL/PLGA NPs;

Figure 5 shows the in-vitro release profile of AZT, AZT-loaded PL NPs, AZTloaded PLGA NPs, AZT-loaded PL/PLGA NPs, and AZT-loaded NPs-in-NFs;

Figure 6 shows the FTIR Spectrum of AZT, raw PVP, PVP-C nanofibers and PVP-M nanofibers;

Figure 7a shows the In Vitro biodegradability of PVP-NFs (PVP-C) and medicated PVP-NFs (PVP-M);

Figure 7b shows the Swelling Profile of PVP-NFs (PVP-C) and medicated PVPNFs (PVP-M);

Figure 7c shows the Water Vapor Permeability of PVP-NFs (PVP-C) and medicated PVP-NFs (PVP-M);

Figure 7d shows the porosity of PVP-NFs (PVP-C) and medicated PVP-NFs (PVP-M);

Figure 8 shows a plot of Cumulative drug permeated (gg/cm²) through excised bovine cornea versus time from the NPs-in-NFs insert compared to AzaSite® eye drops;

Figure 9(a) shows the mucoadhesive/degradability study experiment setup;

Figures 9(b)-9(h) show images of bovine-eyes during the mucoadhesive and degradability experiment;

Figures 9(i)-9(k) show the results of the cell viability study on the unloaded and loaded NFs matrices using a human endothelial cell line;

Figure 10(a-c) show the in-vivo pharmacokinetic study experiment setup (NFs application and aqueous humor sample withdrawal);

Figure 10(d) shows the concentration of AZT in the aqueous humor of rabbit’s eyes versus time (hours) for the NPs-in-NFs insert compared to AzaSite® eye drops; and

Figures 10(e-g) show the microscopic histological investigation of desiccated stained corneal tissues (H&EX200), (h) retina (H&EX200), and (i) ciliary body (H&EX200);

EXAMPLES

Materials

Azithromycin dihydrate was provided by October Pharma, 6th of Ocotber City, Egypt.

Poly (lactic-co-glycolic acid) copolymer (PLGA) was obtained from Purac, Netherlands.

Pluronic F-127 “poly(ethylene glycol)-block-poly(propylene glycol)-blockpoly(ethylene glycol)”, polyvinyl alcohol (PVA, Mw 12,000-14,000 Da), Polyvinylpyrrolidone (PVP, MW 360,000 Da), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), glutaraldehyde, Fluorescein diacetate, dialysis tubing cellulose membrane (molecular weight cut-off 12,000 g/mole), disodium hydrogen phosphate, disodium hydrogen phosphate and dipotassium hydrogen phosphate were purchased from Sigma-Aldrich in China and Germany.

Acetone (99.5%), methanol (HPLC grade) and absolute ethanol (99.5%) were obtained from Piochem, Egypt.

All other reagents were of analytical grade and used as received.

Methods

Characterisation methods • Particle size, polydispersity index and zeta potential of nanoparticles were measured by dynamic light scattering (DLS) with a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) at 25 °C after suitable dilution with purified water.

• Morphological characterization of nanoparticles was performed using a scanning electron microscope (SEM) (Nona Nano SEM, FEI, USA).

• Fourier Transform Infrared (FTIR) spectroscopy on nanoparticles and nanofiber matrices was performed using a FTIR spectrometer (Thermoscientific, USA) in the range of 600 - 4000 cm'¹.

• Differential scanning calorimetry (DSC) analysis on nanoparticles and nanofiber matrices was performed using a DSC (Q20, TA instrument, USA) to evaluate thermal properties of different samples up to 350 °C at a rate of 10 °C/min under nitrogen atmosphere (25 mL/min).

• Thermogravimetric analysis (TGA) on nanoparticles and nanofiber matrices was used to confirm the thermal behaviour of samples using TGA (Q500, Thermoscientific, USA) up to 500 °C of 10 °C/min in air.

• The X-ray diffraction (XRD) patterns on nanoparticles were evaluated by X-ray diffractometer (Philips PW 1390) using Cu Ka1 as an X-ray source.

• The morphology of the nanofibers was assessed using a scanning electron microscope (Nova Nano SEM, FEI, USA) and Image j software was used to determine the nanofiber diameter and polydispersity index.

• The distribution of nanoparticles within the nanofiber matrix was investigated by confocal laser microscopy (CLSM, Nikon, USA) using a Fluorescein diacetate (FDA) -loaded nanoparticles instead of the drug.

Analyses

AZT concentrations in different samples were determined by HPLC (Agilent 1260 system) using a Zorbax C18 column (4.6x250mm, 5pm) with KH₂PO₄ and methanol (90:10, v/v, pH adjusted to 8 using 1M NaOH) as mobile phase and flow rate 1.5 ml/min. The detection was set at 210 nm with 100 μΙ_ injection volume

The entrapment efficiency (EE%) of different nanoparticles was determined directly using HPLC method at 210 nm by dissolving the nanoparticles. EE% was calculated using Eq. (1):

EE% = ((Entrapped AZT)/(Total AZT))x 100 Eq. 1

The in-vitro release profile of AZT from different nanoparticles and nanofibers was studied using the dialysis bag method. Briefly, one ml of AZT-loaded nanosuspensions were placed in a dialysis bag (Mw cut-off 12 kDa; Severa) as donor compartment and immersed in 30 ml PBS (pH 7.4) as receptor compartment at 37°C under mild agitation (50 rpm). Aliquots were withdrawn at predetermined time intervals (0.25, 0.5, 1.5, 2, 4, and 24h, 2, 3, 4, 5 and 7 d) then replaced with fresh PBS. Samples were quantified for AZT using HPLC at 210 nm after appropriate filtration. The concentration was calculated using Eq. (2).

Cn = Cn means + A/V Σ”=ί Cs means (2) where Cn is the expected n^th sample concentration, Cn means is the measured concentration, A is the volume of withdrawn aliquot, V is the volume of the dissolution medium, n-1 is the total volume of all the previously withdrawn samples before the currently measured sample, and Cs is the total concentration of all previously measured samples before the currently measured sample.

Four different kinetic models (zero order, first order, Higuchi, and Korsmeyer-Peppas) were used to fit the release data obtained from different nanoparticles and nanofiber matrices using the excel add-in software package, DDSolver as follows:

Zero order: Q_t = Q_o + K_ot(3)

First order: log Q_t = log Q_o - t / 2.303(4)

Higuchi: Q_f = K_Ht⁰⁵(5)

Korsmeyer-Peppas: log Q_t/ Q„= n logt + log k(6) where, Q_f is the fraction of drug released at time t, Q_o is initial fraction of drug in solution, K_o is zero order release constant, K-ι is first order release constant, K_H is Higuchi dissolution constant. In Korsmeyer-Peppas, Q/Q» is the fraction of drug released at time t, Q» is the total drug released, k is a kinetic constant, and n is the exponent explaining the drug release mechanism.

Biodegradability of the nanofibers was evaluated by suspending pre-weighed nanofiber matrices in PBS, then incubating at room temperature for an hour. The matrices were then withdrawn, dried, weighed and suspended again in the PBS. The weight remaining percentage of the nanofiber matrices were calculated at every time point using Eq. (7).

Reminaing Weight % = (remaining weight/initial weight) x 100 (7)

Swellability of the nanofibers was determined as an indication of their hydrophilicity. Pre-weighed samples were suspended in PBS, withdrawn, dried using a filter paper to remove excess buffer, and then reweighed again at different time points until maximum swelling was obtained. The swelling percentile was calculated using Eq. (8), where Wd is the initial weight of the dry sample and l/l/s is the weight of each swelled sample at certain time point.

Swelling% = ((swelled sample weight - initial weight)/initial weight) x 100 (8)

Water vapour permeability (WVP) of the nanofibers was determined to measure the capability of the ultimate ocular inserts to permit gas flow. The experiment was run according to ASTM E96 desiccant method. A known amount of dried anhydrous silica gel was added to an Erlenmeyer flask whose top was covered with the nanofibers. Then the whole setup was placed in an environment with 75% humidity for 1 hour before calculating WVP of according to Eq. (9), where Δ W is the change in weight of silica gel due to water vapour permeability, A is the surface area of the membrane covering the flask top and At is the change in time.

WVP = ((Difference in silica gel weight )/(Surface area)) X Difference in time (9)

Porosity per volume of the nanofibers was measured using a Pycnometer (Ultrapyc 1200 e, Quantachrome instruments, USA). Samples of known dimensions were weighed and placed in the pycnometer container. Then, equations (10) and (11) were used to calculate the porosity per volume and porosity percentage, respectively.

Porosity per Volume = (1 — sample volume) /(Total Volume) (10)

Porosity % = Porosity per Volume X 100 (11)

Statistical Analysis

All results are expressed in mean ± standard deviation. Significant difference tests were applied like student's t-test and one-way analysis of variance (ANOVA) to all data obtained. All tests were estimated using the software GraphPad Prism Software Version 6

Preparation of Drug-containing Nanoparticles

Azithromycin (AZT) was loaded into single and mixed polymeric nanoparticles, PLGA, PL and mixed PLGA/PL by nanoprecipitation. Briefly, an organic solution, 100 mg AZT in 5 mL of either pure acetone or 95% acetone containing PLGA, PL or PLGA/PL, was slowly poured under moderate stirring into 10 mL of an aqueous solution of either 0.5% PVA aqueous solution or pure distilled water. The nanoparticles were formed spontaneously. Finally, the nanoparticle suspension was stirred for 24 h at room temperature until the complete evaporation of the organic solvent.

In order to optimize the preparation of AZT-loaded nanoparticles, a facecentered central composite design (CCD) was constructed with a combination of two factors (independent variables); ratio of PL to AZT (X1; where 1 and 3 were low and high levels) and ratio of PLGA (X2; where 0 and 4 were low and high levels) at two factorial levels, as set out in Table 1 below. The value for alpha was 1 to fulfil the facecentered design. Physicochemical properties of the produced nanoparticles were the dependent variables: particle size (PS, Y1), polydispersity index (Pdi, Y2), Zeta potential (ZP, Y3), and encapsulation efficiency (EE, Y4), as set out in Table 2. According to the CCD matrix generated by Design Expert® 10.0.1 software (Stat-Ease, Inc., Minneapolis, MN, USA.) a total of 13 experiments were generated. The experimental responses were analyzed using analysis of variance (ANOVA) to identify the significance of the effects and interactions between factors (p-value was <0.05). Software optimization module was used to simultaneously fulfil the predetermined criteria (minimum PS and Pdi and maximum ZP and EE). Finally, a post-analysis was performed to set the factor levels and provide predictions with interval estimates.

Table 1: Values of the two experimental factors and measured responses according to the matrix designed by Face-centered central composite design_________

Factors Measured responses

F#	Space type	PL ratio	PLGA ratio	PS (nm)	Pdi	ZP (mV)	EE%
F1	Factorial	1	0	87.73	0.22	-14.5	29
F2	Factorial	3	0	90.21	0.26	-15.4	57
F3	Factorial	1	4	222.2	0.33	-20.2	60
F4	Factorial	3	4	185.6	0.36	-22	83
F5	Axial	1	2	175.6	0.37	-18.1	55
F6	Axial	3	2	119.9	0.3	-19.6	78
F7	Axial	2	0	66.22	0.24	-15.2	56
F8	Axial	2	4	180.3	0.32	-21.3	76
F9	Center	2	2	106.8	0.31	-19.1	70
F10	Center	2	2	103.1	0.3	-18.7	77
F11	Center	2	2	107.2	0.35	-18.5	77
F12	Center	2	2	105.6	0.37	-19.3	76
F13	Center	2	2	110.4	0.36	-19.9	77

Abbreviations: PL, pluronic; PLGA, poly(lactic-co-glycolic acid); PS, particle size; Pdi, polydispersity index; ZP, Zeta potential; EE, encapsulation efficiency.

Table 2: Coefficient estimate and p-value for that coefficient between experimental factors and measured responses

	Intercept	A	B	AB	ΑΛ2	ΒΛ2
cn	CE	109.09	-14.97*	57.32*	-9.77	32.46*	7.97
Q.	p-value		0.0094	< 0.0001	0.1007	0.0012	0.2409
Q	CE	0.31	1.953E-017	0.048*
Q.	p-value		1.0000	0.0136
Q.	CE	-19.09	-0.7*	-3.06*	-0.225	0.238	0.84*
N	p-value		0.0044	< 0.0001	0.3147	0.3730	0.0122
LU	CE	75.3737	12.49*	12.66*	-1.42	-9.13*	-9.20*
LU	p-value		< 0.0001	< 0.0001	0.3651	0.0013	0.0012
Abbreviations:	CE, Coefficient estimate;	A, PL ratio;	B, PLGA ratio; PS, particle size;

PDI, polydispersity index; ZP, Zeta potential; EE, encapsulation efficiency. * Values were referred to significant coefficient estimate

Table 3: Correlation between experimental factors and measured responses

Factors Measured responses

	PL ratio	PLGA ratio	PS (nm)	PDI	ZP (mV)	EE%
		PS	-0.226 *	0 864 **
Ό CD	(/) φ
Ε 3	(/) c	PDI	0	0.687 **	0.545 **	-
ω	ο
cc φ	Q. (/)	ZP	-0.215 *	-0.941 **	-0.695 **	-0.769 **	-
S	S’
		EE%	0.587 **	0.595 **	0.198 *	0.659 **	-0.788 **	-

* Coefficient values were referred to partial correlation; ** Coefficient values indicate an appropriate correlation.

The results indicated that the dependent variables were significantly affected by all material attributes (Table 1). According to the quadratic experimental model, PS was ranged from 66.22 to 222.2 nm, as shown in Fig. 2a, and was significantly increased (p<0.01, Table 2) with increasing PLGA ratio (correlation coefficient (r) = 0.864, Table 3). Additionally, all cases demonstrated an acceptable homogeneity of particle size distribution with PDI ranging from ~0.2 to -0.4, as shown in Fig. 2b. ZP ranged from 14.5 to -22 mV, as shown in Fig. 2c, with a significant decreasing (p<0.01, Table 2) as PLGA ratio increased (r=-0.941) and PL ratio increased (r=-0.215) as in Table 3. ZP data were correlated negatively with PS (r=-0.695) and PDI (r=-0.769). EE% ranged from 29 to 83%, as shown in Fig. 2d, and increased (p<0.01, Table 2) with increasing PLGA ratio (r=0.595) and PL ratio (r=0.587). Also, EE% increased when particles ZP decreased (r=-0.788).

The Software optimization module was adjusted to minimize PS and PDI and maximize ZP and EE. Based on the data generated, the suggested optimum formulation with PL ratio 2.4 and PLGA ratio 0.3 produced a maximum desirability function of 0.771. The optimized nanoparticles predicted parameters were PS of 89.42nm, PDI of 0.28, ZP of -16.1 and EE% of 62.23%. The predicted outcomes obtained from the model and actual results were compared and revealed a low % bias which confirmed the fitting and validity of the generated mathematical model.

Characterisation

SEM images provided evidence of the successful synthesis of AZT-loaded nanoparticles as shown in Fig. 1a. The SEM results were found to be in good agreement with the results obtained from prediction model of CCD experimental design.

IR spectra of plain drug (AZT), individual AZT loaded PL and PLGA nanoparticles as well as mixed AZT-PL/PLGA nanoparticles were scanned in the range of 1000-4000 cm'¹ as shown in Fig. 2. AZT shows characteristic peaks at 3485 cm'¹ and 3560 cm'¹ that correspond to the OH group stretching and the tightly bound H2O molecules within AZT crystal lattice respectively. In addition, it shows other distinctive peaks at 1720 cm'¹, 1607 cm'¹ and 1453 cm'¹ which correspond to the C=O group stretching, ring C-O-C group stretching and OH group bending. On the other hand, PL NPs show two distinctive peaks at 2881 cm'¹ and 1466 cm'¹ that correspond to the CH2 group stretching and bending respectively. PLGA spectrum shows characteristic peaks at 2949 cm'¹ and 1746 cm'¹ owing to CH₂ stretching and C=O group stretching respectively. The successful formation of AZT-PL NPs, AZT-PLGA NPs and mixed AZT-PL/PLGA NPs was confirmed by the presence of distinctive peaks of both AZT, PL or/and PLGA together as illustrated in the spectrum.

DSC was carried out to detect the success of encapsulation of AZT within the polymer matrices within PL and PLGA as shown in Figs. 3a and 3b. DSC thermograms show the variation in the heat flow within the sample as a function of increasing temperature. DSC thermograms reveal the appearance of a distinctive endothermic peak at 126.95 °C that corresponds to the melting point of free AZT. This peak has disappeared in both AZT-PL NPs and AZT-PLGA NPs DSC charts. This confirms that AZT has been encapsulated successfully within both of the synthesized polymeric nanoparticles.

The thermal properties of the different formulations also have also been investigated through thermogravimetric analysis illustrated in Fig. 3b that shows the variation in the weight loss percentage as a function of increasing the temperature. The ATm and decomposition pattern changed after the incorporation of either AZT within the polymeric nanoparticles matrix or the AZT-loaded PL/PLGA NPs within PVP nanofibers. This confirms the successful development of the target materials. Also, this is with a good agreement of DSC thermograms.

XRD diffraction was carried out to confirm the success of the encapsulation of AZT within the polymer matrices within PL and PLGA as shown in Fig. 4. AZT XRD pattern exhibited sharp multiple peaks (7.92, 9.27, 9.86, 11.99, 15.58, 16.38, 16.87, 18.75, 19.78, 21.62, and 42.97) which represented the crystalline nature of the drug. In the XRD of PL peaks intensities were observed at 2Θ (19.09, and 23.48) while observed PLGA peak was at 19.22. In case of AZT-loaded PL/PLGA NPs showed absences of AZT sharp crystalline peaks, which became too broad without sharp peaks which indicates complete transformation of crystalline AZT into amorphous form. Formation of amorphous AZT is in a good agreement with DSC results.

Analyses

The release profile of AZT exhibited a fast dissolution during 12 h (-80%) and all AZT dissolved after 48 h (Fig. 5). On the other hand, the encapsulation of AZT in PL NPs resulted in a significant controlling of AZT release with -50% released during 12h, then a complete release within 48h. Furthermore, a significant extended release was observed with PLGA NPs as only -65% of AZT released after 10 days. The AZTloaded PL/PLGA NPs release profile was between PL NPs and PLGA NPs as -45%, -60% and -95% released during 12h, 48h and 10 days, respectively.

The incorporation of AZT-loaded PL/PLGA NPs in a nanofiber matrix resulted in a slight delay of AZT release, with -40%, -50% and -90% released during 12h, 48h and 10 days, respectively as demonstrated in Fig. 5. The mathematical modelling was Korsmeyer-Peppas model similar to AZT-loaded PL/PLGA NPs as presented in Table 4.

The release profiles of AZT, loaded NPs and loaded NPs-in-NFs were fitted to different kinetic models. The best fitting model for release of AZT and AZT-loaded PL NPs were first-order and Higuchi model, respectively, with highest r² (Table 4). On the other hand, the best fitting model for release from PLGA based NPs were KorsmeyerPeppas model with highest r². Korsmeyer-Peppas is a model that described drug release from a carrier system. The diffusional exponent (n) is used to describe the drug-release mechanism. All formulations with Korsmeyer-Peppas model exhibited a Fick diffusion model as n value less than 0.5 according to classification previously described.

Table 4: Mathematical models of the regression for in-vitro release profiles of AZT, AZT-loaded PL NPs, AZT-loaded PLGA NPs, AZT-loaded PL/PLGA NPs, and AZTloaded NPs-in-NFs

Model	Equation	AZT	AZT-PL NPs	AZTPLGA NPs	AZTPL/PLGA NPs	AZT NPsin-NFs
Zero-order	F=kO*t	-0.3612	0.3401	0.2112	0.2431	0.1277
First-order	F=100*[1-Exp(-k1*t)]	0.9844*	0.8703	0.4308	0.7984	0.5898
Higuchi	F=kH*tA0.5	0.5451	0.8848*	0.7785	0.8077	0.7460
KorsmeyerPeppas	F=kKP*tAn	0.8572	0.8572	0.9716*	0.9780*	0.9730*
Model parameter	k1 =0.234	kH=12.12 4	kKP=13.3 , n=0.284	kKP=19.1 , n=0.294	kKP=19.0 , n= 0.274

* symbol refers to best fitting model

Preparation of Nanofiber matrix comprising Drug-containing Nanoparticles

AZT unloaded/loaded nanofiber matrices were prepared using 15 %w/v 10 polyvinylpyrrolidone solution. To prepare the drug loaded inserts, a predetermined amount of AZT-loaded nanoparticles was added to PVP solution. Maximum incorporated amount of AZT-loaded nanoparticles within the electrospinning solution was determined upon reaching the most uniform nanofibers produced after optimizing electrospinning parameters which were flow rate, externally applied electric field, 15 distance between needle tip and collector, and speed of the moving spinneret (NANON-O1A electrospinner, MECC, Japan).

Polymer solution was fed in a 5 ml plastic syringe (13.1 mm diameter) and connected to plastic nozzle (1.5 mm diameter) then to 18 gauge stainless steel needle. The optimum electrospinning parameters achieved were found to be 2 ml/hr flow rate, 20 22 kV applied voltage, 15 cm distance between needle tip and receiving collector, and

120 mm/sec speed. The resulting nanofiber matrix dimensions were 120 mm length and 0.5 mm thickness. The temperature and humidity ranges inside the electrospinner were recorded to be 32 - 35 °C and 40% - 50% respectively.

Characterisation

The morphology of the nanofiber matrix, either control PVP-NFs (PVP-C) or drug loaded NPs-in-NFs (PVP-M) was investigated through both scanning electron microscope (SEM) and confocal laser scanning microscope (CLSM). Fig. 1 (c-j) shows the SEM micrographs of each of the nanofiber matrices along with their corresponding histograms and a 3D simulation of both of their upper and lower views.

The SEM image of PVP-C (Fig. 1c) confirms the successful production of control nanofibers with very negligible amount of beads. In addition, its histogram (Fig. 1d) shows that the diameter of the nanofibers ranged from 200 to 550 nm. Fig. 1e-f shows that 3D simulation of the topography of PVP-C NFs at both the upper and level views respectively. It is clear that most of the area is smooth with a very minor abundance of very small beaded regions on both sides.

The SEM image of PVP-M (Fig. 1g) confirms the successful formation of very uniform, free-beaded electrospun nanofibers. The corresponding histogram (Fig. 1h) shows that the nanofiber diameter increased in size compared to the control one. This is attributed to the incorporation of AZT-loaded NPs within the nanofiber matrix. The 3D simulation images of the upper and lower topography of PVP-M (Fig. 1 i-j) confirmed the successful homogenous distribution of AZT-loaded NPs within the PVP matrices.

The morphology of the nanofibers as well as the homogenous distribution of fluorescent dye-Loaded NPs within PVP nanofibers was also confirmed using CLSM as illustrated in Fig. 1b. The 3D model of z-stack images generated by CLSM revealed the distribution of the polymeric nanoparticles within nanofibers, in agreement with the SEM micrographs.

IR spectra of plain drug (AZT), raw PVP powder, PVP NFs (as control) and AZT-loaded NPs in PVP NFs were performed in the range of 800-4000 cm'¹ as shown in Fig. 6. AZT has distinctive peaks at 3485 cm'¹, 3560 cm'¹, 1720 cm'¹, 1607 cm'¹ and 1453 cm'¹. Control PVP nanofibers shows a distinctive peak at 3395 cm'¹ corresponding to the overtone of the C=O group. Also two characteristic peaks appear at 2953 cm'¹ and 2900 cm'¹ that correspond to the ring asymmetric stretching of CH₂ and chain symmetrical CH₂ stretching respectively. In addition, the spectrum shows other three characteristic peaks at 1644 cm'¹, 1494 cm'¹ and 1462 cm'¹ that correspond to the amide group, C=O group and C-N stretching peaks. Through inspecting the spectrum of drug-loaded PVP nanofibers, it could be seen that it shows both distinctive peaks of AZT and control PVP nanofibers which confirms the successful incorporation of AZT-PL/PLGA NPs within the PVP nanofiber matrices.

Furthermore, DSC was carried out to confirm the success of encapsulation of AZT within the polymer matrices (Fig. 3a). DSC thermograms show the variation in the heat flow within the samples as a function of increasing temperature. The distinctive endothermic AZT peak has disappeared in both of the AZT-PL NPs and AZT-PLGA

NPs DSC charts. This confirms that AZT has been encapsulated successfully within both of the polymeric nanoparticles. Additionally, the ATm and decomposition pattern changed after the incorporation of either AZT within the polymeric nanoparticles or the AZT-loaded PL/PLGA NPs within the PVP nanofiber matrices (Fig. 3b). This confirms the successful development of the drug-containing nanoparticles and the nanoparticlecontaining nanofiber matrix.

Analyses

The in-vitro biodegradability study was performed to show that ocular inserts formed from the nanofiber matrix would decompose easily and safely in simulated physiological medium (PBS).

Fig. 7a shows the in-vitro biodegradability of both the control and drug-loaded PVP nanofiber ocular inserts over 60 minutes. It can be seen from the figure that both ocular inserts are highly biodegradable. Figure 7a shows that more than 75% of the ocular insert is degraded only after 1 hour. It can also be seen that incorporation of AZT-PLGA/PL NPs within the nanofiber matrices slightly retards the biodegradability process, presumably due to the presence of the less biodegradable PLGA within the insert composition.

The swelling study was carried out to investigate the degree of hydrophilicity of the materials as well as to understand the AZT release mechanism from the ocular insert. Fig. 7b shows that both control and drug-loaded ocular inserts achieve their maximum swelling after only 30 minutes whereupon their weights were increased by more than 200%. It can also be seen that the drug-loaded ocular insert has slightly decreased swelling due to the abundance of AZT-PL/PLGA NPs.

A water vapour permeability (WVP) experiment was conducted as shown in Fig. 7c to ensure the ocular inserts are able to maintain air perfusion and lachrymal fluids when placed inside the eyes. The results show that both control and drug-loaded ocular inserts possess high porosity and high water vapour permeability. It could also be seen that there is no significant difference between the control and the drug-loaded inserts which suggests that the AZT-Loaded NPs are homogenously distributed within the nanofibers matrices without forming any beads or blocking nanofibers pores.

A porosity test was performed to estimate the porosity percentile among the volume of the ocular insert. High porosity is mandatory to ensure the proper flow of oxygen and lachrymal fluids during the placement of the ocular insert inside the eyes. Porosity percentage of both control and drug-loaded nanofibers were estimated using a pycnometer and displayed as illustrated in Fig. 7d. It could be seen from figure 7d that both control and drug-loaded ocular inserts show no significance difference as both of them proved to possess more than 99% porosity of their volume.

Ex-vivo evaluation

Ex-vivo transcorneal permeation study

An ex-vivo transcorneal permeation study was performed using bovine eyes obtained from a local slaughterhouse. Eyes were collected directly after sacrificing the animals within 1 h. Healthy corneas without any signs of epithelial damage were dissected and carefully collected. The cornea along with the surrounding scleral tissues were carefully excised and washed with normal saline, then stored in PBS. Nanofibers comprising AZT-containing nanoparticles were suspended in 1 ml PBS and placed in glass cylindrical tubes (2 cm in diameter and 10 cm in length) with one end tightly covered with a cornea. The available tissue area for diffusion corresponded to 3.14 cm².The tube was incubated in 30 ml of PBS (pH 7.4) and kept at 37°C ±0.5 with mild agitation (50 rpm). 1 ml aliquots were withdrawn at predetermined time intervals (0.25, 0.5, 1.5, 2, 4, 8 and 24h, 2, 4, 9 and 10 d) and replaced with equal volumes of fresh PBS. Samples were analyzed for drug content by HPLC at 210 nm. This experiment was repeated three times. The results are shown in Figure 8.

The results show that the amount of AZT permeated from drug-loaded NPs-inNFs was significantly (p<0.05) lower than from AzaSite® eye drops. A complete AzaSite® dose was permeated within 24h. On the other hand, AZT-loaded NPs-in-NFs took 10 days to achieve similar result. This confirms the controlled release properties of the nanofiber matrix comprising drug-loaded nanoparticles.

Ex-vivo mucoadhesive/degradability study

The experimental setup was designed for observing the time of degradation and adhesion of nanofiber matrix to bovine-eye. The experimental setup is shown in Fig. 9a. Briefly, bovine-eyes were washed with normal saline, fixed on 100 mL beaker. 1 cm² nanofiber matrix was placed on the bovine eye cornea. A syringe pump (NE-4000, New Era Pump Systems Inc., USA) was adjusted to accurately pump 0.4 mL/h PBS to mimic lacrimal fluid flow rate. A silicone tube was fixed using a clamp to adjust the water dropping above the nanofiber matrix. The degradation and adhesion were evaluated visually at different time intervals. The results are shown in Figures 9b-9h. The NFs showed adhesive property to eye surface.

Cell Viability Study

A cell viability experiment was carried out according to ISO 10993-5 using the MTT assay. Human endothelial cell line (WISH, ATCC®CCL-25™) was used to evaluate the ocular insert biocompatibility. An amount of 200 pl_ of cell suspension (0.5X10⁵ cells) was added to every well containing a sample of nanofibers in a 96 wellplate. Nanofibers along with cells were left in an incubator for 48 hours. Afterwards, MEM with 10 % FBS was used to wash the wells 2-3 times after withdrawal of the medium from the plate. Then an amount of 200 μΙ_ of MTT (5 mg/ml) was added into the wells and left inside a CO₂ incubator for 6-7 hours before adding 1 ml of DMSO and mixing it well. The optical density of the viable cells was detected by assessment of the purple developed due to the presence of viable cells and formazan crystals. The absorbance of the cell suspension was detected at 595 nm using a spectrophotometer and DMSO as a blank. The percentage of cell viability was estimated using Eq. 12.

Cell Viability % = ((Mean Optical Density)/(Control Optical Density)) x 100 (12)

The results confirm that the NFs samples are nontoxic when compared to the negative control using human endothelial cell line. No significant difference in cell viability % was noted between the unloaded and drug loaded NFs. Furthermore, it was observed that cells were attached to the tested NFs matrices (Fig. 9j-k).

In-vivo Evaluation

In-vivo pharmacokinetic study

All experiments were approved by the Institutional Ethics Committee at Misr University for Science and Technology in compliance with the ARRIVE guidelines and ARVO guidelines for the use of animals in Ophthalmic Research. Six albino rabbits (2-3 kg) with clinically normal eyes were allocated in the study. A nanofiber comprising drug-containing nanoparticles insert was instilled in the lower cul-de-sac of the eyes then the both eyelids were gently closed for 10 s. At time intervals of 0.5, 1, 2, 4, 8, 12 and 24h, 2, 4 and 7 d, samples of aqueous humor were removed from the anterior chamber of each eye after local anesthetic instillation using 4% xylocaine solution topically.

100 μΙ samples and 100 μΙ methanol was mixed together and kept in a refrigerator for 1 h to precipitate proteins and then centrifuged at 5000 rpm for 15 min. AZT was measured using the previously mentioned HPLC method considering matrix effect of aqueous humor. The pharmacokinetic parameters including the maximum concentration of the drug in the aqueous humor (Cmax), time required to reach the maximum concentration (tmax), area under the concentration-time curve (AUC₀-t) and half-life of drug were calculated using the predefined functions of PKSolver add-in program. The results are shown in Table 5 and in Figures 10a-10c.

Both Cmax and AUC_t were significantly increased (p<0.05) in AZT-loaded NPsin-NFs compared to AzaSite® (Table 5 and Fig. 10d). The increases were 1.6-fold and 14.8-fold respectively. Furthermore, the nanofibers T_max was 6-fold longer than AzaSite®. Also, a 72.7 h residence time achieved by nanofibers whereas a residence time of 9 h was achieved with AzaSite®.

Table 5: Pharmacokinetic parameters of AzaSite® and AZT-loaded NPs-in-NFs in aqueous humor of rabbit’s eye

Treatment	Cmax (gg/ml)	Tmax (h)	AUCt (μ£.ΙΐΓ/ιη1)	MRTt(h)	Ti/2(h)
AzaSite®	26.0 ±3.2	8.0	336.8 ± 39.8	9.0	6.3
AZT-NPs-in-NFs	43.2 ±5.1	48.0	4988.7 ± 588	72.7	93.5

In-vivo tolerance study

An acute tolerance test was performed on three albino rabbits (2-3 kg) weight to investigate the ocular tolerance of the nanofiber comprising drug-containing nanoparticle insert.

Healthy animals were housed singly in standard cages, in a light-controlled room at 24±4°C, with no food or water restriction. A single dose nanofiber comprising drug-containing nanoparticle insert was instilled in one eye, and the other was kept as control. After 7 days, rabbits were euthanized by overdose of sodium pentobarbital given intravenously via ear vein. Cornea, iris and retina of each rabbit were isolated, washed by physiological saline then fixed in 10% formalin for 24 h. The dehydration of different specimens was done using serial dilutions of alcohol (methanol, ethanol and absolute ethanol) and water. Paraffin beeswax tissue blocks were prepared by embedding specimens in paraffin at 56°C in hot air oven for 24 hrs. A section of 4 pm thickness was prepared by microtome. The obtained sections were stained by hematoxylin and eosin (H&E) stain after deparaffinization, and then examined by light electric microscope 45. For Grading of stained specimens, five grades were used to describe obtained specimens according to Ximenes et al 46. Specimens with grade I oedema (mild) presented only a slight and sparse separation between structures. Cases where the separation was more frequent and widespread were classified as grade II oedema (mild-moderate). Grade III oedema showed generalized swelling (moderate). In grade IV oedema (moderate-severe), the separation was wider and very frequent. Finally, specimens with grade V oedema (severe) presented greater separation with generalized swelling.

The corneal tissue specimens of the control untreated group showed normal histological structure which consisted of outer non-keratinized stratified squamous epithelium with intact epithelial basal lamina and Bowman’s layer. The corneal stroma appeared as regularly collagen lamellae with intact Descemet’s membrane which lined by single layer of flat endothelial cells (Fig.lOe). In the treated group with nanofiber comprising drug-containing nanoparticles, after one week the collagen lamellae were separated and the spaces contain lightly staining material in-between fibrous bundles indicating mild-moderate oedema (grade II) (Fig. 10f). Furthermore, corneal stroma showed mild oedema (grade I) with slight, sparse separation of stromal structures after two weeks of treatment (Fig. 10g).

In both groups, the retinal layers showed normal histological structure during the experimental period. The retinal pigment epithelium; rods and cones (photoreceptors); external limiting membrane; outer nuclear layer; outer plexiform layer; inner nuclear layer; inner plexiform layer; ganglion cell layer; nerve fiber layer and inner limiting membrane appeared without any pathological alterations (Fig. 10h). Also, the ciliary body is the anterior portion of the uveal tract, which is located between the iris and the choroid showed normal histological structure with normal loose vascular and pigmented connective tissue that contained bundles of ciliary muscle. Its inner surface was lined by outer pigmented and inner non-pigmented cell layers and the inner surface highly folded forming the ciliary processes (Fig. 10i).

Claims (23)

CLAIMS:

1. An ocular drug delivery system comprising a nanofiber matrix, wherein said nanofiber matrix comprises drug-containing nanoparticles.

2. A system as claimed in claim 1, wherein said nanoparticles are impregnated in said nanofiber matrix.

3. A system as claimed in claim 1 or 2, wherein said nanoparticles have an average diameter of 30-450 nm.

4. A system as claimed in any preceding claim, wherein said nanoparticles comprise a biodegradable, hydrophobic 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, 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

5. A system as claimed in claim 4, wherein said biodegradable, hydrophobic polymer is poly(lactic-co-glycolic acid) (PLGA).

6. A system as claimed in any preceding claim, wherein said nanoparticles comprise a biodegradable, amphiphilic polymer selected from block copolymers of ethylene glycol-propylene glycol-ethylene glycol (PEG-PPG-PEG), polylactic acidethylene glycol-polylactic acid (PLA-PEG-PLA), poly(lactic-co-glycolic acid)-ethylene glycol-poly(lactic-co-glycolic acid) (PLGA-PEG-PLGA) and polyglycolic acid-ethylene glycol-polyglycolic acid (PGA-PEG-PGA).

7. A system as claimed in any preceding claim, wherein said nanoparticles comprise a mixture of at least one biodegradable, hydrophobic polymer and at least one biodegradable, amphiphilic polymer.

8. A system as claimed in any preceding claim, wherein said drug is selected from an anti-microbial drug, an anti-inflammatory drug, an anti-glaucoma drug, an analgesic, an anaesthetic or combinations thereof.

9. A system as claimed in any preceding claim, wherein said nanoparticles comprise 5-50 wt% of drug, based on the total weight of the nanoparticles.

10. A system as claimed in any preceding claim, wherein said nanofibers have an average diameter of 60-3950 nm.

11. A system as claimed in any preceding claim, wherein said nanofibers comprise a mucoadhesive polymer.

12. A system as claimed in claim 11, wherein said mucoadhesive polymer is selected from polyvinylpyrrolidone, copovidone, copolymers of substituted vinyl pyrrolidone, polyvinyl alcohol, polyacrylic acid, cellulose polymers, chitosan, chitosan derivatives, polyethylene glycols (PEG), polypropylene glycols (PPG) and natural gums.

13. A system as claimed in claim 11 or 12, wherein said mucoadhesive polymer is polyvinvylpyrrolidone.

14. A system as claimed in any preceding claim, wherein said nanofibers comprise 1-50 wt% of nanoparticles, based on the total weight of the nanofiber matrix.

15. A system as claimed in any preceding claim, wherein said nanofibers comprise 0.5-30 wt% of drug, based on the total weight of the nanofiber matrix.

16. A system as claimed in any preceding claim in the form of an ocular insert, preferably a patch.

17. A system as claimed in any preceding claim, wherein said system provides controlled-release of drug over a period of at least 3 days.

18. A method of making an ocular drug delivery system as claimed in any one of claims 1 to 17 comprising:

(ii) preparing nanofibers impregnated with drug-containing nanoparticles, preferably by electrospinning; and (iii) forming the resulting nanofibers into said ocular drug delivery system.

19. A method as claimed in claim 18, further comprising the step of:

(i) preparing drug-containing nanoparticles, preferably by nanoprecipitation.

20. An ocular drug delivery system for use in the treatment of ocular diseases.

21. A system as claimed in claim 20, wherein said ocular disease is selected from glaucoma, blepharitis, corneal tear/injury, dry eye, aphakia, pseudophakia, astigmatism, blepharospasm, cataract, conjunctival diseases, conjunctivitis, allergic conjunctivitis, corneal diseases, corneal keratitis, corneal ulcer, dry eye syndromes, eyelid diseases, lacrimal apparatus diseases, lacrimal duct obstruction, myopia, presbyopia, pupil disorders, refractive disorders, strabismus, acute macular neuroretinopathy, Behcet's disease, choroidal neovascularization, diabetic uveitis, histoplasmosis, infections such as bacteria, fungal or viral infections, macular degeneration (e.g., acute macular degeneration, non-exudative age related macular degeneration and exudative age related macular degeneration), edema (e.g., macular edema, cystoid macular edema and diabetic macular edema), multifocal choroiditis, ocular trauma which affects a posterior ocular site or location, ocular tumors, retinal disorders such as central retinal vein occlusion, diabetic retinopathy, proliferative diabetic retinopathy, proliferative vitreoretinopathy (PVR), retinal arterial occlusive disease, retinal detachment, uveitic retinal disease, sympathetic ophthalmia, Vogt Koyanagi-Harada (VKH) syndrome, uveal diffusion, a posterior ocular condition caused by or influenced by an ocular laser treatment, posterior ocular conditions caused by or influenced by a photodynamic therapy, post-surgical inflammation (cataract, glaucoma, corneal transplant), photocoagulation, radiation retinopathy, epiretinal membrane disorders, branch retinal vein occlusion, anterior ischemic optic neuropathy, nonretinopathy diabetic retinal dysfunction, and retinitis pigmentosa.

22. Use of a drug in the manufacture of an ocular drug delivery system as claimed in any one of claims 1 to 17 for the treatment of ocular diseases.

23. A method of treating an ocular disease in a patient in need thereof, comprising:

(i) placing an ocular drug delivery system as claimed in any one of claims 1-17 into the eye of said patient; and

5 (ii) maintaining said system in said eye for a therapeutically effective period of time.