Characterization of a novel nanomicellar formulation for ocular delivery of Cyclosporine A

University of Pisa

Department of Pharmacy

Corso di Laurea Magistrale a Ciclo Unico

In

Chimica e Tecnologia Farmaceutiche

Graduation Thesis

Characterization of a novel nanomicellar formulation for ocular delivery

of Cyclosporine A

Supervisors Candidate

Dr. Daniela Monti Chiara Marchetti

Prof. Patrizia Chetoni

Abstract

The aim of the present study was to evaluate a combined strategy involving nanomicelles based on non ionic surfactants and hyaluronic acid (HA) as mucoadhesive polymer to improve ocular bioavailability of cyclosporine A, a poorly soluble drug.

From a previous study, a nanomicellar formulation, Nano1HAB-CyA, containing a total

surfactant amount of 1% w/w (Vitamin E-TPGS and IGEPAL® in a ratio of 2.25:1), hyaluronic acid 0.01%w/w, and loaded with CyA (0.1%w/w) was selected by a statistical design of experiment (DoE). Nanomicelles were prepared by the melting method and using an isotonic phosphate buffer solution (PBS) as solvent.

First of all, the technological characterization of the prepared formulation was performed by measuring the pH, osmolarity and size distribution immediately after preparation and filtration through a 0.22 m cellulose acetate membrane filter to assess the ocular biocompatibility and the appropriate dimension to reach the anterior chamber of the eye, respectively. pH and osmolality of the formulation resulted suitable for a topical ocular administration and the particle diameter was in a size range of 10-18 nm, allowing to penetrate the ocular tissues. Afterwards, the drug content (entrapment) and the loading capacity of the formulation was determined by reverting the nanomicelles in organic solvent to extract the drug from the nanomicelles core and the CyA concentration was then analysed by RP-HPLC. The drug entrapment and loading efficiency was in a range of 75 – 85% and 10 – 12%, respectively, with 0.1%w/w final CyA concentration. Subsequently, short-term stability of nanomicelles at 4, 20 and 32°C (storage, room and ocular surface temperature) was also evaluated measuring the amount of drug remained in the formulation at predetermined time interval (1,2,7,14 and 30 days) by HPLC, and assessing drug possible degradation and checking nanomicelles structural preservation by dimensional and turbidity analysis. CyA was stable at 4°C and 20°C up to 30 days with no sign of degradation; the nanomicelles seem to maintain their structure over the time at 4°C and 20°C until at least 48 hours. At 32°C the nanomicelles lose their structure and regenerate in approximately 11 min when allowed to cool at room temperature. Moreover, a comparison between the formulation under study and the same formulation without HA (Nano1-CyA) was done to evaluate the HA role as a stabilizer in addition to its mucoadhesive properties. No statistical differences between the two nanomicellar formulations were observed in term of size (Nano1HAB-CyA vs Nano1-CyA), even if Nano1-CyA showed a higher coefficient of variation

formulation Nano1HAB-CyA were performed at 32°C in water by using the dialysis method

following the process for 30 hours. An ethanolic solution of the drug at the same concentration (0.1% w/w) was used as reference solution. Before the in vitro release experiments, the possible interactions between the polypeptide and the materials used in this test have been determined. On the basis of the obtained results, all materials used for the release studies were subjected to treatment with a Tween® 20 (0,05%) solution. The Nano1HAB-CyA formulation showed a diffusive release profile, releasing in 6 hours twice the

amount of CyA (50 g) compared to the reference solution (25 g) suggesting a positive influence of the nanomicellar structure on the release of the drug. Moreover, the release of Nano1HAB-CyA was followed up to 30 hours showing that the formulation under study was

able to release a total amount of drug of about 114 g.

Furthermore, an ATR-FITR analysis on raw materials (CyA, hyaluronic acid, vitamin E-TPGS and IGEPAL®) and on the freeze-dried Nano1HAB-CyA formulation was carried out to

evaluate probable structural changes and interactions among the formulation components. In this study, it is to be highlighted that the Nano1HAB-CyA formulation ATR-IR spectrum

exhibits CyA characteristic peak, which could demonstrate the successful loading of the drug in the nanoparticles, and a C=O vibration signal shift of Vit E-TPGS, suggesting an interaction between the drug and the surfactant.

Finally, the in vivo pharmacokinetics of the formulation under study in the tear fluid of New Zealand rabbits (animal model) was evaluated in order to investigate if the nanomicellar formulation produced a sustained release in the precorneal area, avoiding daily repeated administrations. As reference, a formulation with the same composition of an US commercial product not available on the European market (Restasis®_{), and loaded with the same}

amount of CyA of the nanomicelles formulation, was used. Pharmacokinetics studies have shown that Nano1HAB-CyA and the reference emulsion were comparable concerning the

pharmacokinetics parameters: elimination rate constant (Ke), half-life (t1/2) and medium tear

Table of Contents

I. Introduction………... 1

1. The Anatomy and Physiology of the Eye……….…..… 2

2. The Ophthalmic Therapy………... 6

2.1 The Topical Administration……….………... 7

3. Topical Ophtalmic Drug Forms……….………... 10

3.1 Novel Ocular Drug Delivery Systems………..……… 13

4. Nanomicelles………..……… 15

4.1 Surfactant Nanomicelles………..……….. 15

4.2 Polymeric Nanomicelles………... 16

4.3 Methods of Nanomicelles Preparation……….… 17

5. Nanomicelles in Ocular Drug Delivery………... 19

5.1 Application in the Ocular Anterior Segment……… 19

5.2 Application in the Ocular Posterior Segment……….. 21

II. Experimental Section……….. 24

1. Aim……….………... 25

2. Materials and Methods……….……… 27

2.1 Materials………..………... 27 2.1.1 Cyclosporine A…...……….. 27 2.1.2 Vitamin E TPGS………...……… 28 2.1.3 IGEPAL® CA-630……… 28 2.1.4 Sodium Hyaluranate……… 28 2.2 Methods……….... 29

2.2.1 Preparation of CyA-Loaded Nanomicelle Formulation.………... 29

2.2.2 Preparation of CyA-loaded Emulsion………. 31

2.2.3.1 Size Distribution……… 31

2.2.3.2 Osmolality and pH………..………...….…. 32

2.2.3.3 Entrapment and Loading Efficiency…...………... 32

2.2.3.4 Stability Studies………..………... 32

2.2.4 In Vitro Drug Release Studies………... 33

2.2.5 ATR-FTIR Analysis……… 34

2.2.6 In Vivo Studies……… 34

2.2.6.1 Animals……… 34

2.2.6.2 Evaluation of the pharmacokinetic in the precorneal area……….. 35

2.2.7 Analytical Method of CyA……… 35

3. Results and Discussion.……… 37

Table………..…. 42

Figure………... 45

1. The Anatomy and Physiology of the Eye

Figure 1 - The anatomy of the eye.

The eye is an isolated sensory organ and is located in the front upper part of the skull. The outer tissues of the eye consist in three layers. The outermost layer consists of the cornea and sclera (fibrous tunica), which provide protection for internal structures. The middle coat, the uveal tract, is a vascular layer, which has a nutritive function and is composed by choroid, ciliary body and the iris. The innermost layer is the retina (nervous tunica) containing photoreceptors and is involved in the reception of visual stimuli. The inner eye is divided by the lens, which separates the aqueous and vitreous humours; instead, the iris separates the aqueous humour into the anterior and posterior chambers. A brief description of the eye structures is shown below starting from the outside:

a. The Precorneal Tear Film. The precorneal tear film is a very thin layer, which is continuously wetting the corneal epithelium, the conjunctiva and the walls of the conjunctival cul-de-sac. The tear film has many functions, i.e., provides a smooth uniform refractive surface, lubricates the ocular surface, protects against bacterial infection, and removes cellular debris and foreign matter. This moisturizing action is important not only for the maintenance of a clear cornea but also for the eyelids movement.

The tear film is composed by three layers: lipid, aqueous, mucin coats. The outermost layer is the lipid layer which is approximately 0.1 m thick and is composed by wax and

cholesterol; its function is to reduce the evaporation from the underlying aqueous phase and, in this way, preventing the cornea from drying out. The aqueous layer lies below the lipid layer and is the larger component of the tear film (6-10 m thick), consisting of watery lacrimal secretion provided by the numerous accessory lacrimal glands, most of which are situated in the upper conjunctival fornix. The innermost layer is the mucin coat, which is secreted by conjunctival goblet cells and glands of Manz. Mucin is involved in adhesion of the aqueous phase to the underlying corneal epithelium and acts as a wetting agent reducing the interfacial tension between corneal epithelium and tears (Saettone et al, 1999).

The water-soluble elements of the tears are electrolytes, proteins and peptides such as albumin, lysozyme, lactoferrin, cytokines, growth factors etc (Lemp, 2008).

The tear film in the eye is constantly replenished and this process is connected to the blinking, which sweeps the excess fluid by the nasolacrimal duct. The lacrimal fluid in humans has a volume of 7 L with pH 7.4 (Shahwal, 2011).

b. The Cornea. The cornea is an avascular, transparent structure composed of five layers with a total thickness of 300 – 500 m. These layers include: epithelium, Bowman’s membrane, stroma, Descemet’s membrane, and endothelium.

The epithelium plays a critical role in the maintenance of a barrier against tearborne agents and in a balanced stromal hydration. It is composed of five to seven cell layers, with a total thickness of 35-50 m. The cells at the base are columnar but, as they are squeezed forwards by new cells, they become flatter identifying three groups of cells: basal cells, an intermediate zone of 2-3 layers of polygonal cells (wing shaped) and squamous cells. The permeability of the intact corneal epithelium is low, suggesting that tight junctions exist between the cells of the outer layer. The outer layer of the surface cells possess microvilli on their anterior surface maybe to anchor the precorneal tear film. The cells of the basal layer show extensive lateral interdigitation of plasmatic membranes and are, therefore, relatively permeable. Immediately adjacent to the epithelium there is a less ordered region of the stroma, the Bowman’s membrane followed by stroma, a hydrated (75-78% water) matrix of collagen fibrils and glycosaminoglycans (GAGs), which constitutes 90% of the corneal thickness. As a purely hydrophilic structure, the stroma can act as barrier for very lipophilic substances. This arrangement is followed by Descemet’s membrane and corneal endothelium. Descemet’s membrane forms the basement membrane for the corneal

endothelium. Corneal endothelium is a monolayer of polygonal cells, most of which are hexagonal in shape with about 20 m diameter and 4-6 m thickness. These cells play a key role in maintaining corneal transparency through their transport, synthetic, and secretory functions (Sunkara and Kompella, 2003). The endothelium is in contact with aqueous humour of anterior chamber, and it is crossed by a passive flux of water towards the stroma while an active pump mechanism generates a flux in the opposite direction controlling corneal turgescence.

c. The Conjunctiva. The conjunctiva is a thin, transparent mucous membrane lining the inside of the eyelids and continuous with cornea. The conjunctiva can be divided into three layers: (a) an outer epithelium, a permeability barrier, (b) substantia propria, containing nerves, lymphatics, and blood vessels, and (c) submucosa, which provides a loose attachment to the underlying sclera (Sunkara and Kompella, 2003). The conjunctiva, lining the lids, is vascular (palpebral conjunctiva) while on the globe is transparent (bulbar conjunctiva). The area between the lids and the globe is named conjuctival sac.

d. The Sclera. Sclera is the white or opaque fibrous tissue that forms the external protective coat. It is composed, mainly, of collagen fibers, which have a haphazard arrangement that causes the opaque scleral effect (Boddu et al., 2013). Other elements present in the sclera are proteoglycans, elastin, proteins and cellular components and water. The main function of this tissue is to provide a protective layer for the internal components of the eye.

e. The Anterior Chamber and Aqueous Humor. The anterior chamber varies in depth and is bordered by cornea in the front and the pupil and iris diaphragm in the back. It is filled with aqueous humour produced by the ciliary epithelium in the posterior chamber. Aqueous humour is formed from blood plasma by mechanism of diffusion, ultrafiltration and active transport. The ciliary processes of the human eye produce aqueous humour at a rate of 2-3 L/min. Before the aqueous humour reaches the anterior chamber, it flows through the posterior chamber (Boddu et al., 2013). Furthermore, the aqueous humour production and the intraocular pressure are maintained by membrane transport processes (Sunkara and Kompella, 2003).

f. The Iris – Ciliary Body. The iris, ciliary body and choroid comprise the vascular uveal coat of the eye. The anterior iris is immersed in the aqueous humour, which enters the iris stroma through openings or crypts along its anterior surface. The iris receives its blood supply from the major arterial circle, which lies in the stroma of the ciliary body near the iris root. The ciliary body can be divided into the following regions: nonpigmented ciliary epithelium, pigmented ciliary epithelium, stroma, and ciliary muscle. The main arterial blood supply to the ciliary body is through the long posterior and the anterior ciliary arteries (these capillaries are fenestrated and leaky) (Sunkara and Kompella, 2003).

g. The Lens - The lens is a transparent tissue, consisted of 65% water and the rest mainly by proteins. Anteriorly, the lens is in contact with the pupillary portion of the iris, and posteriorly it fits into a hollow depression of the anterior vitreous surface. The major components of the lens are capsule, epithelium, and lens fiber cells. The lens capsule is acellular, transparent, elastic, and acts as an unusually thick basement membrane that encloses the epithelium and lens fiber cells. Membrane transport proteins in the lens play an important role in cell volume regulation, nutrient supply, and lens transparency. All cells of the lens are interconnected by gap junctions (Sunkara and Kompella, 2003).

h. The Retina. The retina is mainly divided in two layers: neural retina (inner layer) and retinal pigment epithelium (RPE - outer layer). The neural retina comprises rods, cones, bipolar cells and ganglionic cells, which are involved in the conversion system of the light into electrical impulses that provides for the normal vision. RPE is a non-visual portion located between the neural section of the retina and choroid and consists of a layer of melanin–containing epithelial cells. Melanin helps in absorption of stray light rays entering the eyeball and prevents the reflection of scattering of light. The RPE forms a blood-retinal barrier through tight junctions that enable the neighbouring cells to connect each other. These cells regulate the trans-epithelial transport of various molecules through apical tight junctions, which retard the diffusion through the paracellular spaces. The numerous microvilli on the neural retinal section help the absorption of various nutrients and thereby maintain the viability of the neural retina. RPE expresses certain efflux pumps, which block the entry of xenobiotics from the extravascular space of the retina (Boddu et al., 2013).

i. Vitreous humour. The vitreous humour consists in a viscous gel-like structure and represents the 80% of the total volume of the eye. This fluid fills up the space between the lens and the retina. Vitreous humour is composed by water (99%), hyaluronic acid, hyalocytes, inorganic salts, sugar, ascorbic acid and a network of collagen fibers. The network of non-branching collagen fibers with hyaluronic acid imparts viscosity to the vitreous humour (Boddu et al., 2013). While the aqueous humour is continuously replenished, vitreous humour has a lower turnover. It serves as a mechanical buffer for the surrounding tissues, exerts an important role in the transmission of the light to the retina, and is involved in the maintenance of the intraocular pressure (Gajraj, 2012).

2.The Ophthalmic Therapy

Topical therapy is the most commonly used for treatment of diseases of the anterior segment of the eye such as ocular surface diseases (conjunctivitis, dry eye, keratitis, etc.), glaucoma and anterior uveitis. These dosage forms like eye drops and suspensions are convenient and easy to instil but suffer from inherent drawbacks that lead to an ocular low bioavailability of drug due to, for example, the tear turnover, poor corneal permeability, nasolacrimal drainage, systemic absorption connected to side effects. To overcome some of these problems and obtain an effective therapy, we can consider drug delivery systems, able to control drug release, both to prolong therapeutic activity and interact better with the ocular barriers or to reach specifically the drug’s site of action.

However, topical therapies are limited for treating disorders of posterior segment due to the greater diffusional distance as well as anatomical and physiological barriers in the eye. In this case intravitreal drug injections have been explored for delivering drugs to target tissues in the eye at therapeutic concentrations.

2.1

The Topical Ocular Administration

Figure 2 - Model Showing precorneal and intraocular events following topical ocular administration of a drug.

The formulation intended to be administered to the eye surface may act either on the precorneal area or penetrate through the ocular structures and be effective into the interior of the eyeball. First of all, there are some anatomical and physiological constrains to take into account when we use a topical administration such as cul-de-sac limited volume, the solution drainage, lacrimation, tear turnover, drug spillage for blinking, conjunctival-scleral absorption, enzymatic metabolism, and protein binding.

The ocular bioavailability remarkably depends on a suitable contact time with the cornea. Instilled solution drainage away from the precorneal area (within 5 minutes after instillation in humans) has been shown to be the most significant factor reducing the permanence of drug in this area. The natural tendency of the cul-de-sac is to reduce its fluid volume to 7-10 L because of the blinking. A typical ophthalmic dropper delivers 30 L, but, most of the active is rapidly lost through nasolacrimal drainage immediately after administration. This drainage mechanism may then cause the drug to be systemically absorbed across the nasal mucosa or the gastrointestinal tract leading to important side effects. The tear turnover acts to remove drug solution from the conjunctival cul-de-sac. Normal human tear turnover is approximately 16% per minute. Moreover, absorbed drug may set off the eye through the canal of Schlemm or via absorption through the ciliary body of suprachoroid into the episcleral space. In addition, even if the drop’s proteinaceous content (albumin, globulins, lysozyme) is very low (about 0.7%), the continuous tear turnover produces new proteins, most of which can bind the drug molecules. Enzymatic metabolism

may contribute to further loss from both precorneal area and cornea.

The lacrimation (tear production), which leads to a dilution of the pharmaceutical form, also influences the ocular drug absorption. It can be induced by many factors such as the type and amount of drug, pH and tonicity of the dosage form, use of adjuvants.

All these factors may influence the ocular drug bioavailability that has been estimated to be 1-2% (or less) of the instilled dose (Macha et al., 2003).

Another important factor is the absorption of the drug into the conjunctiva and sclera, the so-called conjunctival/scleral pathway. Drug loss through this route of absorption may be significant. Conjunctival-scleral pathway is fairly permeable to hydrophilic and large molecule and may serve as a route of absorption for some larger molecules such as proteins and peptides (Urtti, 2006).

The mechanism of permeation of the drug through ocular tissues to arrive into internal structure of the eye is the passive diffusion following the Fick’s first law:

J = - D 𝑑𝐶

𝑑𝑥

where J is the flux rate through the membrane; D is the diffusion coefficient that depends on physic-chemical properties of the drug such as molecular size; dC/dx is the concentration gradient, i.e., the ratio between the difference of concentration to the two sides of the barrier and the thickness of the barrier. Another parameter that influences the flux of the drug through the ocular tissue is the partition coefficient (K), which indicates the affinity of the drug for the lipophilic or hydrophilic compartments. Equilibrium between the two characteristics (lipophilic or hydrophilic) is essential to promote the transocular permeation. K can be experimentally determined using as solvent a mixture of octanol (lipophilic characteristics) and water (hydrophilic portion).

To reach the anterior chamber of the eye, the drug, topically administered, has to pass through the corneal layers. Corneal epithelium, the first barrier, can be crossed though two pathways: transcellular (through the cells) and paracellular (between the cells) pathway (Fig. 3). Lipophilic molecules and molecules with specialized transport processes prefer the transcellular route, whereas hydrophilic molecules prefer the paracellular route (Shahwal et al, 2011).

active transport, and endocytosis. The most of the drugs, no such transporters or receptors exist, are transported by passive diffusion through the apical membrane and across the basolateral membrane to move across the cell interacting with some components of the cell membrane. Physiochemical properties of the drugs that may influence drug penetration via the transcellular pathway, purely lipophilic, include: (1) the lipophilicity of the drug and its octanol-water partition coefficient; (2) its pKa (dissociation constant), which determines the amount of absorbed drug depending on its ionized or unionized form at a given pH; (3) molecular size.

Transport through the paracellular pathway is passive and only limited by the size and charge of the intercellular spaces. It is an aqueous route involving the diffusion of the solute between adjacent epithelial cells/endothelial cells restricted by the presence of the tight junctions or zonula occludens (ZO) (Sunkara and Kompella, 2003).

For most drugs, the multicellular layered corneal epithelium presents the greatest barrier to penetration while stroma and endothelium offer little resistance. Indeed, for too much lipophilic drugs, the corneal stroma represents a rate-limiting barrier to access the inner eye and in this case it may act as a reservoir from which the drug will be slowly delivered to the aqueous humor. Finally, the moderate lipophilic corneal endothelium does not offer significant resistance to drug absorption (Ye et al., 2013). However, considering this anatomic conditions, the drug should have both lipophilic and hydrophilic characteristics to across the different layers of cornea (Shahwal et al, 2011).

In addition to the classical corneal pathway, there is a competing and parallel route of absorption via the conjunctiva and sclera, the so-called conjunctival/scleral pathway; comparing the corneal route with this absorption pathway, it results that the second route is a minor absorption pathway, even though for some compounds its contribution can become significant.

Ahmed and Patton (1985, 1987) investigated corneal versus noncorneal penetration of topically applied drugs in the eye. They demonstrated that noncorneal absorption could contribute significantly to intraocular penetration. Drugs can bypass the anterior chamber and distribute directly to the uvea and vitreous. For drugs with low corneal affinity, such as inulin, this absorption route may be particularly important. In another study, Ahmed et al. (1987) evaluated in vitro the barrier properties of the conjunctiva, sclera, and cornea comparing the diffusion characteristics of various β-blockers. Permeability through the sclera of these compounds was higher than through the cornea related to their

physicochemical properties. The results suggested that the pathway of absorption might be in part influenced by lipophilicity and that hydrophilic compounds seem to prefer the conjunctival/scleral route. Schoenwald et al. (1997) verified that a compound with a remarkable lipophilicity as rhodamine B did not pass through conjunctival/scleral barrier.

Figure 3 - The cornea. Cellular organization of various transport limiting layers.

3. Topical Ophthalmic Drug Forms

The pharmaceutical forms more commonly used in ophthalmic field can be classified on the basis of the physical form in:

• Liquid dosage forms: solutions and suspensions;

• Semisolid dosage forms: ophthalmic ointments and gels; • Solid dosage forms: ocular inserts.

Although many techniques of instilling drugs to the eye have been experimented, the use of eye drops remains the main method of administration for the topical ocular route. The two major physical forms of eye drops are aqueous solutions and suspensions. The solution offers the potential of greater assurance, of uniformity of dosage and bioavailability and simplifies large-scale manufacture. Furthermore, the solution is more easily administrable

with an excellent patient compliance even if it has, as fundamental drawback, the low bioavailability due mainly to the rapid and extensive precorneal loss, the high absorption via the conjunctiva and the nasolacrimal duct leading to systemic side effects. When the drug is not sufficiently soluble in hydrophilic vehicle a suspension can be formulated. A suspension may also be desired to improve stability, bioavailability or efficacy. Most of steroid anti-inflammatory agents (such as prednisolone acetate, dexamethasone) are formulated in ophthalmic suspensions . The critical element of these formulations is the size of particles. Micronized particles (size <10 μm) are accepted in ophthalmic field to not cause irritation of sensitive ocular tissues and to help ensure that a uniform dosage is delivered to the eye. In any case there are a lot of constraints connected to preparation and administration of a stable suspension, i.e., they need to be adequately shaken before use to ensure the correct dosing and reach a moderate increase in bioavailability; and also the suspensions are expensive in terms of production costs.

To increase the ocular contact time and subsequently improve ocular bioavailability of the drug, high molecular weight polymers can be added to the ophthalmic solutions and suspensions raising their viscosity. The most commonly used hydrophilic polymers in ocular field are polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP), and cellulose derivatives such as hydroxypropyl methylcellulose (HPMC), methylcellulose (MC) and polyacrylic acids (carbopols) (Shahwal et al., 2011).

There are many examples on the market of eyedrops containing rheological modifiers such as Fluaton®_{, containing PVA, Flarex}® _{containing HEC, Flumetol}® _{containing a mixture}

of PVA and HPMC, Retaine® _HPMCTM _{lubricant eye drops containing HPMC.}

There is a direct relationship between viscosity and bioavailability up to a plateau after which further increase in viscosity produces only slight or no increase in therapeutic effect. Many research works have demonstrated this correlation and led to the conclusion that ophthalmic liquid formulations should have viscosity in the range of 25 to 50 cP for a greatest permanence in the precorneal area. The addition of a 0.5% PVA solution to eye drops increases the viscosity to about 3 cP (water viscosity= 1 cP) and decreases the drainage rate of topically applied eye drops as compared with aqueous solution. The drainage rate decreases by a factor of 2-3 at viscosities up to 7.7 cP and further increase in viscosity from 7.7 to 75.8 cP is also associated with a decrease by a factor of 2-3. The rate of drainage decreases with additional PVA up to a maximum of 3%, after which there is no longer any advantage (Kebler et al., 1991).

Redkar et al. (2000) evaluated the influence of the viscosity of new possible commercial artificial tears on increasing the permanence time of the formulation in the precorneal area and on producing a stable tear film. MOISOL® _{eye drops (0.3% HPMC) was}

used as reference. As viscosity-imparting agents, HPMC and Dextran 70 were used individually or in combination. An aqueous formulation containing dextran 70 (0.1% w/v) and HPMC (0.7% w/v) appeared the best to maintain for a long time a stable tear film. The experimental formulation with a viscosity of 25.49 cP produced a longer permanence in the precorneal area of the eye drops (2h) than the reference MOISOL which has a viscosity of 42.30cP and a precorneal permanence time of 1,5 h. This seems to confirm that high viscosity is not necessarily correlated with a high bioavailability.

In addition to their thickening effect, many aforementioned polymers possess mucoadhesive properties, i.e. ability to establish adhesive, non covalent bonds with mucin layers coating corneal-conjunctival epithelium, giving ophthalmic drug delivery systems longer times of contact with the absorbing tissues. For an efficient mucoadhesive activity, the polymers need to have one or more of the following features: strong hydrogen binding group, strong anionic charge, high molecular weight, sufficient chain flexibility, surface energy properties favouring spreading onto the mucus. The combination of mucoadhesion and viscosity is a target for an optimal activity (Saettone et al, 1999).

The main semisolid dosage form used in ophthalmology is anhydrous ointment with a petrolatum base or a mixture of petrolatum and lanolin derivatives. Ophthalmic ointments containing antibiotics are used quite frequently following operative procedures. The reduction of dilution of medication via the tear film, resistance to nasolacrimal drainage, and increased precorneal contact time lead to a remarkable increase of bioavailability, whereas the blurred vision, and the discomfort by the patient are disadvantages that limit its use to a night treatment.

An aqueous semisolid gel base has been developed to provide significantly longer residence time into the cul de sac. The gel consists of a high molecular weight crosslinked polymer to provide high viscosity and optimum rheological properties for prolonged ocular retention. Only a relatively low concentration of polymer is required, so that the gel base is more than 95% water.

In-situ gelling systems are viscous polymer-based liquids that exhibit sol-to-gel phase transition on the ocular surface due to change of specific physic-chemical parameter (electrolyte composition, the change in temperature, pH). This kind of formulation has many

advantages, i.e., an easy instillation, a prolonged residence time of the formulation on the surface of the eye (due to gelling) and an easy, reproducible, accurate administration of a dose compared to the application of performed gels.

The ophthalmic inserts are solid devices designed to deliver the drug in the conjunctival cul-de-sac or in the posterior segment of the eye. The main objective of ophthalmic inserts is to maintain longer the formulation in the precorneal area ensuring a sustained release suited for topical or systemic treatment, with an improved bioavailability.

3.1 Novel Ocular Drug Delivery Systems

In the last few decades, new approaches have been used in research for the treatment of ocular diseases, including the nanotechnologies to prepare formulations for drug delivery both in the anterior and in the posterior segment of the eye. Nanosystems are used to increase, on one hand, the aqueous solubility and, on the other hand, the stability of active principles thus improving the ocular tissue compatibility and bioavailability. Several types of nanocarriers for ocular drug delivery were developed, such as liposomes, nanoparticles, nanomicelles and dendrimers, some of which have shown promising results to improve ocular bioavailability. (Figure 4)

Liposomes are lipid vesicles consisting of an outer phospholipid bilayer and an aqueous inner core. They are usually made with phosphatidylcholine, stearylamine, and various amounts of cholesterol or lecithin and 𝛼-L-dipalmitoylphosphatidylcholine. Size varies from 10 nm to 1 m or greater, depending on the type. In fact, they are classified as small unilamellar vesicles (10-100 nm), large unilamellar vesicles (100-300 nm) and multilamellar vesicles, containing more than a bilayer of phospholipids. For ophthalmic applications, liposomes represent the ideal delivery system because of its excellent biocompatibility, its ability to encapsulate both hydrophobic and hydrophilic drugs and its similarity to the cellular membrane structure. However, they are not as stable as polymer based systems and their large-scale production is expensive and very difficult from a technological point of view.. Nanoparticles are colloidal carriers usually composed of lipids, proteins, natural or synthetic polymers. They can be divided in nanospheres, where the drug is uniformly distributed throughout polymeric matrix, and nanocapsules, where the drug is encapsulated in a polymeric shell. Advantages of these nanocarriers are their small size and consequent low irritation, larger dissolution area and greater penetration capacity in eye structures. However, they have limitations related to low loading capacity and rapid elimination, requiring often an

association with mucoadhesive polymers to improve precorneal residence time.

Dendrimers are branched, star-shaped polymeric systems having a size between 2-20 nm. The polymeric structure or dendrimers can create electrostatic or covalent bonds with the drug attached to the surface, thanks to the presence of many functional groups (carboxyl, hydroxyl and amine). During the synthesis process, the size and shape of the dendrimer can be controlled, so the final macromolecule will have a specific “architecture” and specific terminal groups. Their highly branched structure allows attachment of multiple drug molecules, increasing drug loading compared to linear polymers, and, moreover, their spherical shape and ability to penetrate cellular membranes makes them an ideal drug delivery system.

Nanomicelles are a common type of carrier consisting of amphiphilic molecules used to formulate insoluble drugs into a clear aqueous solution, with high bioavailability for the ocular tissues. (Patel et al., 2015, Baranowski et al., 2014, Kalomiraki et al., 2015).

4. Nanomicelles

Nanomicelles are mainly used to prepare aqueous clear solutions of drugs that are insoluble in water. Nanomicelles consist of amphiphilic molecules (surfactants or polymers) that self-assemble to form an organized supramolecular structure in the aqueous mean. They can have different sizes (10-1000 nm) and shapes (spherical, cylindrical etc..), depending on the molecular weights of the components. Nanomicelles self-assembly occurs only above critical micellar concentration (CMC). They consist of a hydrophobic core and a hydrophilic corona, soluble in water (Figure 5), allowing the encapsulation of hydrophobic molecules and remarkably increasing the lipophilic drug water solubility by the formation of hydrophobic interactions together with Van der Waals interactions and hydrogen bonding.

Figure 5- Schematic illustration of formation of spherical micelle and drug encapsulation

Nanomicelles used for ocular drug delivery are usually classified in polymeric, surfactant and polyionic complex (PIC) micelles where the type of carrier is selected on the basis of physicochemical properties of the drug, interactions between drug and surfactant or drug and polymer, the site of action, rate of drug release, biocompatibility and physical stability. Moreover, nanomicelles are an excellent ocular drug delivery system thanks to their nanoscale size, their ability to encapsulate and solubilize hydrophobic drugs producing a clear formulation and enhancing penetration through ocular tissues with minimal or no irritation (Vaishya et al., 2014).

4.1 Surfactant Nanomicelles

Surfactants are amphiphilic molecules with hydrophilic head and hydrophobic tail. Hydro-philic head can be charged (anionic or cationic), zwitterionic or non-ionic. Sodium dodecyl sulfate (SDS, anionic surfactant), dodecyltrimethylammonium bromide (DTAB, cationic sur-factant), ethyleneoxide (N-dodecyl; tetra, C12E4), Vitamin E-TPGS (d-alpha tocopheryl

PEG 1000 succinate), octoxynol-40 (non-ionic surfactants), and dioctanoyl phosphatidyl choline (zwitterionic surfactants) are the most common surfactants used to prepare nanomi-celles. The tail of surfactants is usually a long hydrocarbon chain and rarely includes halo-genated or oxyhalo-genated hydrocarbon or siloxane chain. Micelles are formed when surfac-tants are dissolved in water at concentration above the CMC and also a balance between the various interactions (Van der Waals, hydrophobic, steric etc…) is required for their suc-cessful formulation. Nanomicelles formation is the result of two phenomena: a) Self-associ-ation of surfactants tails due to hydrophobic interactions and b) repulsive forces (steric and electrostatic interactions) between the hydrophilic heads that avoid phase separation. A cor-rect balance between these forces contributes to reduce free energy of the system, due to the removal of lipophilic fragments from the aqueous environment and the re-establishment of hydrogen bond network in water, allowing nanomicelles formation. An important parame-ter is the aggregation number which represents the average number of surfactant monomers in each micelle. Since the nanomicelles formation depends on the non-covalent aggregation of surfactant individual monomers, these structures can exist in different shapes (spherical, cylindrical or planar) and size, which are both influenced by total surfactant concentration, pH, temperature and ionic strength (Cholkar et al., 2014, ).

4.2 Polymeric Nanomicelles

Polymeric nanomicelles consist of amphiphilic polymers having two distinct segments, one hydrophobic that self-assembles to form the core and another hydrophilic that forms instead the corona (Figure 6). Micelles are formed when the individual polymer chains (unimers) are directly dissolved in aqueous solution above the CMC but also above a certain temper-ature called critical micellar tempertemper-ature (CMT). Amphiphilic di-block (hydrophilic-hydropho-bic) or tri-block (hydrophilic-hydrophobic-hydrophilic) copolymers are most commonly used to prepare self-assembled polymer micelles. Polymers used, usually poly(lactide), poly (pro-pylene oxide) (PPO), poly(glycolide), poly(lactide-co-glycolide), and poly(𝜖-caprolactone) (PCL), should be biodegradable and/or biocompatible, allowing the elimination of the inac-tive polymer from the ocular tissues. In particular, poly (ethylene glycol) (PEG) is the most used hydrophilic segment due to its excellent water solubility and biocompatibility. They are characterized by a low CMC and an excellent kinetic and thermodynamic stability in solution compared to surfactant micelles. In fact, the rate of dissociation of unimers (single polymer chains) from polymeric micelles is slower, making the micelles kinetically stable. Thermody-namic stability is achieved by interactions of core-forming blocks as well as the ability of

hydrophilic block to solubilize the supramolecular structure. (Vaishya et al., 2014, Batrakova et al.,2006).

Fig.6- Schematic illustration of polymer micelle formation. Amphiphilic copolymers self-assemble to have a hydrophobic core and a hydrophilic corona structure in aqueous environment.

4.3 Methods of nanomicelles preparation

The most commonly used methods to prepare nanomicelles are direct dissolution, solvent evaporation, film hydration and dialysis method. Encapsulation efficiency in the micelles core depends on the preparation method and on the extent of polymer (or drug-surfactant) interactions. Generally, methods like solvent evaporation and film hydration result in a higher encapsulation of drug.

Direct dissolution is the simplest and most frequently employed method for micelles preparation, starting from copolymers with relatively high water solubility. This method consists of dissolving the drug and copolymer block directly in deionized water or buffer. Stirring, heating and/or sonication may be required in order to load the drug into nanomicelles. This method is employed for moderately hydrophobic polymers such as poloxamers and also to formulate polyionic complex micelles (PICM).

The dialysis method is frequently employed when using amphiphilic polymers with low water solubility and also with drugs soluble in organic solvents such as dimethylsulfoxide (DMSO), N,N-dimethylformamide (DMF), acetonitrile (ACN), tetrahydrofuran (THF), acetone or dimethylacetamide. Copolymer and drug are dissolved in the same organic solvent and the formation of nanomicelles happens by the addition of water to the mixture drug-copolymer.

Nanomicelles are then dialyzed against water for a predetermined period of time to eliminate organic solvent. The choice of the solvent and an appropriate water/organic solvent ratio influences physical properties, size, stability and the efficiency of encapsulation of the drug in nanomicelles prepared. In fact, in a work of Yokoama et al. (1998), the use of DMF rather than DMSO for the preparation of nanomicelles loaded with an insoluble anticancer drug resulted in an increased drug loading efficiency.

In the case of solvent evaporation technique, drug and copolymer are dissolved in the same organic solvent or in a mixture of two miscible solvents. A drug-copolymer film is formed after stirring and drying the mixture of solvents and, then, hydrated with warm water or buffer to get nanomicellar dispersion. The reconstituted sample can be sonicated or passed through a high pressure extruder to obtain an homogeneous dimensional distribution. To avoid phase separation during the evaporation process, a solvent, which both drug and copolymer are soluble in, should be selected.

Another method involves the dissolution of drug and copolymer in a mixture of aqueous and organic solvent followed by lyophilization. The freeze-dried mixture can be reconstituted to obtain drug-loaded nanomicelles. Dimethylacetamide and tert-butanol have been generally employed as co-solvents because of their high vapour pressure that allows rapid sublimation.

Nanomicelles preparation method can influence considerably the physicochemical properties of the final product and drug encapsulation efficiency whereas the size and the polydispersity index depends on the nature of the organic phase, the order of solvent addition and the concentration of the copolymer in the organic solvent (Cholkar et al., 2014).

Figure 7- Micelle preparation methods: (1) simple dissolution (2) dialysis, (3) oil in water emulsion

(4) solvent evaporation and (5) lyophilization or freeze drying.

5. Nanomicelles in ocular drug delivery

5.1 Application in the ocular anterior segment

Many researchers have studied the use of nanomicelles for the delivery of small molecules and several genes to the anterior chamber of the eye. Polymeric and surfactant nanomi-celles have been reported to facilitate penetration through ocular tissues, consequently, im-proving bioavailability. Gupta et al. (2000) developed polymeric nanomicelles composed of copolymers of N-isopropylacrylamide (NIPAAM), vinyl pyrrolidone (VP) and acrylic acid (AA) cross-linked with N, N’-methylene bis-acrylamide (MBA) loaded with ketorolac. No damage was observed on rabbit cornea during permeation studies and bioavailability resulted dou-bled with respect to an aqueous suspension containing the same amount of ketorolac. Civiale and co-workers (2009) used copolymers of polyhydroxyethyl-aspartamide (PHEA) with side chains containing polyethylene glycol (PEG) or hexadecylamine (C16) to prepare dexamethasone (DEX) loaded nanomicelles for ocular delivery. In particular, micelles were prepared from PEG-C(16) and PHEA-PEG-C(16). In vitro permeability studies on rabbit

conjunctival and corneal epithelial cells showed an increased permeation of DEX nanomi-celles when compared to solutions and suspensions of the same drug. In vivo studies pro-duced similar results, suggesting nanomicelles as potential carriers for ocular drug delivery. Pepic et al. (2010), have carried out a study on a chitosan micellar system, taking advantage of the mucoadhesive properties of chitosan. Micellar formulations were composed of poly-oxyethylated nonionic surfactant Pluronic F127 (F127) and chitosan (CH) and loaded with DEX. This study demonstrated that CH was able to enhance in vitro DEX release, improving ocular bioavailability since the area under the curve (AUC) values were 2-4 fold increased in bioavailabilty after the administration of DEX micellar system compared to a reference DEX suspension.

In another study, Di Tommaso et al. (2011) developed polymeric micelles based on methoxy poly(ethylene glycol)-hexylsubstituted poly(lactides) (MPEG-hexPLA) for topical ocular de-livery of Cyclosporine A (CyA), a poorly soluble drug. Nanomicelles, with a mean diameter of 35 nm and a CyA concentration of 0.05% w/v,.were prepared by a solvent evaporation method. Excellent ocular biocompatibility, transparency and stability of these micelles was observed both in vitro and in vivo, suggesting the use of this CyA formulation as possible eye drop formulation.

A CyA nanomicellar solution based on a non ionic surfactant of poly(ethylene glycol)- fatty alcohol ether type (Sympathens AS, 0.3% w/v) was developed also by Luschmann et al. in 2013. The average size of the micelles, loaded with 0.05% w/v of CyA, was in the range of 9.7-10.1 nm and showed no sign of ocular irritation when administered. The na-nomicellar solution exhibited higher levels of CyA in the cornea (826±163 ng), than that obtained with a cationic emulsion (750 ng/g) and Restasis (350 ng/g). Therefore, nanomi-celles of CyA could be used for treatment of inflammatory corneal diseases, improving the patient compliance and reducing daily administration.

Then Vadlapudi et al. (2013) developed a clear, aqueous nanomicellar formulation of bioti-nylated lipid prodrug (biotin-12Hydroxystearic acid-acyclovir, B-12HS-ACV) for the treat-ment of corneal herpetic keratitis, using a mixture of Vitamin E TPGS and octxynol-40. TEM analysis suggested that nanomicelles (average size of 10.78 nm) were spherical and ho-mogenous with no presence of aggregates. A sustained release of B-12HS-ACV from the nanomicellar formulation was observed for a period of 4 days, compared to 100% release of B-12HS-ACV in ∼6 h from its ethanolic solution.

Moreover, topical gene delivery through ocular administration is very promising for the treat-ment of a wide range of corneal diseases such as corneal neovascularization, dry eye syn-drome, corneal scarring, corneal angiogenesis and inflammation. For example, Liaw et co-workers (2001) managed to transfer plasmid DNA with LacZ gene in vivo with poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) non ionic polymeric mi-celles and deliver it to the corneal epithelium.

5.2 Application in the ocular posterior segment

Topical surfactant aqueous nanomicellar formulation can also be employed to non-inva-sively deliver therapeutic agents to posterior ocular tissues. The most common diseases which can cause loss vision are the age related macular degeneration (AMD), uveitis, cho-roidal neovascularization (CNV), diabetic retinopathy (DR) and posterior vitreoretinopathy (PVR). Strategies that have been employed so far to deliver the drug via oral or intravenous pathway may not reach the desired therapeutic concentration in the site of action due to the different barriers that drug must overcome. On the other hand, local ocular delivery strate-gies such as intravitreal, retrobulbar and suprachoroidal administration are associated with numerous side effects. For this reason, attempts were made to drive therapeutic agents to the back of the eye including that of taking advantage of nanocarriers as nanomicelles. Ideta et al. (2005) performed studies to deliver fluorescein isothiocyanate-labeled poly-L-lysine (FITC-P(Lys)) into the choroid to treat CNV. The molecule was encapsulated into polyethylene glycol-block-poly-α,β-aspartic acid micelles, resulting in polymeric ionic (PIC) nanomicelles . The nanomicellar formulation was administered in vivo in rats after inducing the disease, using free FITC-P(Lys) as control. Blood and retina-choroid levels and tissue distribution were determined following intravenous administration. The formulation showed a Cmax at 4 hours in retina-choroid with a residence time of 168h. Rats that received free

FITC-P(Lys) died within one hour while no death occurred in animals treated with the mole-cule encapsulated into PIC micelles. This could demonstrate that PIC micelles accumulated in the CNV lesions, reaching therapeutic concentrations in the posterior segment of the eye. Mitra et al. (2011) reported application of nanomicelles for delivery of drugs in the posterior segment of the eye via topical administration. At the beginning, voclosporin was encapsu-lated in micelles consisting of a mixture of Vitamin E-TPGS and Octoxynol-40 as surfactants. The same procedure was followed with rapamycin and dexamethasone (DEX) obtaining in any case a size in the range of 10-25 nm. Efficacy of voclosporin nanomicellar formulation

(0.2%) for the treatment of canine keratoconjunctivis sicca was compared with a CyA oint-ment (Optimmune) through Schirmer test, a measure of tear production, and corneal obser-vation. Schirmer test values for the nanomicellar formulation were above the normal value (> 15 mm/min) while control values remained below the threshold. Moreover, tolerability studies were performed against Restasis (a CyA commercial emulsion) in New Zealand rab-bits, showing that the nanomicellar solution was well tolerated with significant reduction of the ocular irritation. High concentrations of voclosporin were observed in the back of the eye in contrast with minimal levels found in aqueous humour, lens and vitreous humour. In fur-ther studies, attempts were made to non-invasively deliver DEX and rapamycin to the pos-terior tissues of the eye with the nanomicellar formulation. The solubility of DEX and rapamy-cin was increased of 6.7 and 1000 times, respectively, and moreover, high levels of both drugs in retina-choroid were determined, while minimal levels were found in the anterior chamber, suggesting a non-corneal route of drug absorption to the posterior segment. In conclusion, nanomicellar formulations could be employed to deliver therapeutic agents to the posterior ocular tissues via topical instillation, providing a good tolerability and reaching therapeutic concentrations.

Figure 7- Schematic representation of back of the eye drug delivery for drugs entrapped in mixed nanomicelles

1. Aim

The main goal of this thesis is the development of a new nanocarrier drug delivery system to deliver Cyclosporine A (CyA) in the precorneal area for the treatment of the dry eye syn-drome. More specifically, the aim of the present study was to evaluate a combined strategy involving nanomicelles based on non ionic surfactants and hyaluronic acid (HA) as muco-adhesive polymer to improve ocular bioavailability of cyclosporine A, a poorly soluble drug. From a previous study, a nanomicellar formulation, Nano1HAB-CyA, containing a total

sur-factant amount of 1% w/w, hyaluronic acid 0.01%w/w, and loaded with CyA (0.1%w/w) was selected by a statistical design of experiment (DoE). This nanomicellar system should easily circumvent constraints that usually affect the administration of insoluble drug in the precor-neal area (low bioavailability, ocular irritation, redness, inflammation, blurred vision, etc…) and, at the same time, should prolong the drug residence time in the precorneal area by taking advantage of the mucoadhesive properties of hyaluronic acid.

Nanomicelles were prepared by melting at 50°C appropriate amounts of Vitamin E-TPGS and IGEPAL® as surfactants, cyclosporine A and hyaluronic acid. Afterwards, an isotonic phosphate buffer solution (PBS) preheated at 40°C was added to the melted mixture to obtain the final formulation.

First of all, the technological characterization of the prepared formulation was performed by measuring pH and osmolarity to assess the ocular biocompatibility. Size distribution was determined by dynamic light scattering immediately after preparation and filtration through a 0,22 m cellulose acetate membrane filter. The drug content (entrapment) and the loading capacity of the formulation was determined by reverting the nanomicelles in organic solvent to extract the drug from the nanomicelles core and the CyA concentration was then analysed by RP-HPLC.

Short-term stability of nanomicelles at 4, 20 and 32°C (storage, room and ocular surface temperature) was also evaluated measuring the amount of drug remained in the formulation at predetermined time interval (1,2,7,14 and 30 days) by HPLC, and assessing drug possible degradation and checking nanomicelles structural preservation by dimensional and turbidity analysis. Regeneration time was also checked when the nanomicelles structure was lost. Moreover, a comparison between the formulation under study and the same formulation without HA (Nano1-CyA) was done to evaluate the HA role as a stabilizer in addition to its mucoadhesive properties.

Secondly, in vitro release studies of CyA from the nanomicellar formulation were performed at 32°C in water by using the dialysis method following the process for 30 hours. An etha-nolic solution of the drug at the same concentration (0.1% w/w) was used as reference so-lution. Before the in vitro release experiments, the possible interactions between the poly-peptide and the materials used in this test have been determined. On the basis of the ob-tained results, all materials used for the release studies were subjected to treatment with a Tween® 20 (0,05%) solution.

Furthermore, an ATR-FITR analysis on raw materials (CyA, hyaluronic acid, vitamin E-TPGS and IGEPAL®) and on the freeze-dried Nano1HAB-CyA formulation was carried out

to evaluate probable structural changes and interactions among the formulation compo-nents.

Finally, the in vivo pharmacokinetics of the formulation under consideration in the tear fluid of New Zealand rabbits (animal model) was evaluated in order to investigate the capacity of the nanomicellar formulation to provide a prolonged release in the precorneal area, avoiding daily repeated administrations. As reference, a formulation with the same composition of an US commercial product not available on the European market (Restasis®), and loaded with the same amount of CyA of the nanomicelles formulation, was used.

2. Materials and Methods

2.1 Materials

The following products were used:

• D-alpha tocopheryl polyethylene glycol 1000 succinate, VE-TPGS (Vitamin E TPGS, Eastman Chemical Company, Kingsport, Tennessee);

• 4-(1,1,3,3-Tetramethylbutyl)phenyl-polyethyleneglycol or Octylphenoxy poly(ethyleneoxy), OPPEE (IGEPAL® CA-630, Sigma-Aldrich, Milan)

• Sodium Hyaluranate, HA (Chemofin s.r.l, Milan)

• Cyclosporine A, CyA (Poli Industria Chimica s.p.a., Milan)

• Polyethylene glycol sorbitan monolaurate, Polyoxyethylenesorbitan monolurate, (Tween® 20, Sigma-Aldrich, Milan)

• Polyethylene glycol sorbitan monooleate, Polyoxyethylenesorbitan monooleate or Polysorbate 80 (Tween® 80, Sigma-Aldrich, Milan)

• 1,2,3-Propanetriol, Glycerin, Glycerol (Sigma-Aldrich, Milan) • Castor Oil (Sigma-Aldrich, Milan)

• Acrylates/C-10-30 Alkyl Acrylate Crosspolymer, (Carbopol® 1342, Biochim s.r.l., Milan)

• All other chemicals and solvents were of analytical grade.

2.1.1 Cyclosporine A (CyA)

Cyclosporine A (CyA) is a cyclic undecapeptide of 1202.6 Da obtained by an extract of soil fungi. It is a powerful immunosuppressant with a specific action on T-lymphocytes and it was first developed to counter graft rejection following organ transplantation. Its high molecular weight and hydrophobic nature (Log P=1.4 to 3.0; solvent dependent) are responsible for CYA low water solubility (6.6 to 106 μg/mL; temperature dependent). CyA also showed in-teresting activity in uveitis, corneal healing, vernal keratoconjunctivitis and other inflamma-tory diseases of the eye. In the last few years, because of its very poor water solubility,

formulation strategies have focused on developing novel systems to deliver the solubilized drug to the corneal surface (Lallemand et al., 2017).

Fig.1 Structure of CyA.

2.1.2 Vitamin E TPGS (VE-TPGS)

D-α-tocopheryl polyethylene glycol 1000 succinate (Vitamin E TPGS) is a water-soluble derivative of natural Vitamin E, which is obtained by esterification of Vitamin E succinate with polyethylene glycol (PEG) 1000. It has an average molecular weight of 1,513 Da and presents an amphiphilic structure with a lipophilic alkyl tail and hydrophilic polar head; its hydrophilic/lipophilic balance value is 13.2 and it has a relatively low critical micelle concentration (CMC) of 0.02% w/w. Vitamin E-TPGS is a waxy solid with a melting point of about 37-41°C (Guo et al., 2013). As well as bulky structure and large surface area, it can be chosen like a safe pharmaceutical adjuvant such as absorption enhancer, emulsifier, solubilizer, and stabilizer (Wu et al, 2015).

Fig.2 Structure of Vitamin E-TPGS

2.1.3 IGEPAL® CA-630 (OPPEE)

IGEPAL® CA-630 (Octylphenoxy poly(ethyleneoxy)ethanol, OPPEE) is a liquid, viscous, nonionic surfactant, having a HLB of 13.4 and a CMC of 0.08 mM. It is chemically indistin-guishable from Nonidet P-40 and is similar to Octoxynol 40.

Fig.3 Structure of Igepal®

2.1.4 Sodium Hyaluranate

Hyaluronic acid (HA) is a naturally occurring linear polysaccharide composed of alternating residues of the monosaccharides D-glucuronic acid and N-acetyl-D-glucosamine linked in repeating units. It is most frequently referred to as hyaluronan because it exists in vivo as a polyanion and not in the protonated acid form. Hyaluronic acid is widely distributed in the extracellular matrix of connective tissues and it is present in synovial fluid, in the aqueous and vitreous humour of the eye and other tissues (Goa and Benfield, 1994). HA has been used in topical ophthalmic formulations due to its viscoelastic and mucoadhesive properties. Numerous studies have reported that HA prolonged the ocular residence time and the bio-availability of many drugs in solution form by binding to the layer of natural mucin on the

corneal epithelium (Liao et al., 2005). Furthermore, HA has other beneficial effects on the corneal epithelium, including reduction of healing time, lubrication of the ocular surface and protection against dehydration caused by the inflammatory response (Salzillo et al., 2016).

Fig.4 Structure of HA

2.2. Methods

2.2.1 Preparation of CyA-Loaded Nanomicelle Formulation

Cyclosporine A-loaded surfactant nanomicelle formulation was obtained by direct melting method. The hydrophobic core of polymers and drug (0.1%w/w) interact together promoting the drug entrapment in the nanomicelles core. Therefore, nanomicelles were obtained by mixing Vitamin E-TPGS (69.23 mg) and OPPEE (30.77 mg) (2.25:1.0 weight ratio) and CyA (15.0 mg) was added to the surfactant mixture. At this point, 200 mg of a sodium hyaluronate (HA) dispersion (0.5%w/w) was added to the blend and the final mixture kept at 50°C in a water bath until complete surfactant melting. To obtain 10 ml the final nanomicellar solution (Nano1 HAB-CyA), an isotonic phosphate buffer solution (PBS, pH=7,4, 66,7 Mm),

preheated to 40°C, was added to the melted mixture. The total percentage of surfactant and HA in the final formulation was of 1.0 % and 0.01% w/w, respectively. The mixture was kept under stirring overnight at room temperature and protected from the light. The solution obtained was finally filtered through a 0,22 m filter to remove the unentrapped drug aggregates and other foreign particulates. The nanomicelles without drug (Nano1 HAB) and without HA (Nano1-CyA) were prepared to use as control.

2.2.2 Preparation of CyA-loaded emulsion

A CyA emulsion with the same composition of an US commercial product not available on the European market (Restasis®) was prepared, in order to be used as control in pharmacokinetic studies in rabbits. 10 ml of two emulsions loaded with 0.05% and 0.01% of CyA were prepared by dissolving the drug in castor oil (1.25%). This lipophilic phase was then heated at 65°C and added to an aqueous solution containing Tween® 80 (1.00%), glycerine (2.2%) and deionized water at the same temperature. Afterwards, a carbomer 1342 (0.05%) dispersion, maintained at 65°C, was added to the previously obtained mixture and immediately neutralized with 50 l of sodium hydroxide to adjust pH favouring the gel formation. The preparation was kept under stirring until cooling and formation of a stable milky emulsion.

2.2.3 Characterization of the nanomicellar formulations 2.2.3.1 Size Distribution

The mean hydrodynamic diameter of the nanomicellar formulation prepared was determined by dynamic light scattering (DLS, Beckman Coulter® N4 Plus, Beckman Coulter s.r.l, Milan, Italy), immediately after preparation. DLS is a non-destructive technique, which allows measure the molecule/particle size in submicrons level, using a He-Ne laser at a wavelength of 632.8 nm. The instrument consists of a mechanic-optical component for the detection and an electronic component for control and data processing (N4 Plus Software). The samples were analysed in polystyrene cells. Before the analysis, the instrument was set in term of viscosity (1.002 CP), refractive index (1.333) of the diluent (water), temperature (20°C). The concentration of the formulation was chosen in order to be within the range of 5x10⁴ and 1x10⁶ counts per second (cps). Dynamic light scattering measurements were done on all the nanomicellar formulations before any further analysis to confirm the diameter of nanomicells prepared. The instrument was set with the following parameters:

Scattering angle: 90° Run time: 200 s

Equilibration time: 3 minutes

Range of measurement: 1-5000 nm, bins 31

The SDP (Size Distribution Processor) analysis was used since it gives the most accurate particle-size distribution for complex systems with more size populations. The algorithm

used in SPD analysis is based on the CONTIN program. All measurements were performed six times on the nanomicellar formulations immediately after preparation

2.2.3.2 Osmolality and pH

All prepared formulations were examined for osmolality and pH. Osmolality values were determined by a freezing point depression osmometer (5R version, Roebling, Germany) and pH values were determined by a digital pH-meter (611 model, Orion Research, USA). All measurements were performed six times on the nanomicellar formulations immediately after preparation.

2.2.3.3 Entrapment and Loading efficiency

The entrapment efficiency is the percentage of drug loaded with respect to the amount of initial drug (Cholkar et al., 2014). The total amount of entrapped drug in the formulation was determined by RP-HPLC. The filtered CyA-loaded nanomicellar formulation was diluted 1:200 with acetonitrile to release the CyA in the surrounding organic environment due to the formation of reverse micelles, which the orientation of hydrophobic and hydrophilic seg-ments are reversed in and the CyA amount in the diluted sample was analysed. The percent entrapment and loading efficiency of CyA were calculated according to Eqs. 1 and 2, re-spectively.

(1) Percent drug entrapped = mass of CyA in nanomicelles /mass of CyA initial in the formulation *100

(2) Loading Efficiency= mass of CyA in nanomicelles/ mass of CyA + mass of polymers in the formulation *100

2.2.3.4 Stability studies

At the beginning, thermal stability of Nano 1 HAB – CyA was carried out keeping the formu-lation at 4, 20 and 32°C (storage, room and ocular surface temperature) and determining size distribution and turbidity after 24 and 48 hours. Samples at 4°C and at 20°C were kept in fridge and at room temperature, respectively, while samples at 32°C were kept in a ther-mostated water bath. Particles size was determined by dynamic light scattering using the method described previously. Turbidity analysis was done by measuring absorbance with

an UV/Visible spectrophotometer (Shimadzu UV-2101PC), using PBS as blank. 500 l of each sample was placed in a cuvette and absorbance was measured in a range of  of 400 and 600 nm.

After determining the temperature at which the clear nanomicellar solution becomes turbid, the formulation was allowed to cool down to room temperature. The time required for the turbid solution to become transparent again was recorded as regeneration time (R.T). Then, different samples of the formulation were stored at 4°C (fridge) and 20°C (room tem-perature) at 1, 2, 7, 14 and 30 days. The concentration of CyA in the samples was analysed by HPLC and the CyA recovery (%) in respect of the initial amount was calculated. To verify the role of HA on the stability of nanomicelles, the nanomicellar size distribution of

Nano1HAB-CyA was compared with that of same formulation without HA (Nano1-CyA) after

1, 2, 3, 6 and 7 days using dynamic light scattering as described previously.

Size distribution data of all experiments were tested for statistically significance difference using the one-way analysis of variance (ANOVA) (GraphPad Software Inc., La Jolla, CA). Data are the average of at least six determinations ± standard deviation (SD). Groups that were calculated with p value lower than 0.05 (p<0.05) were considered to have statistical difference.

2.2.4 In Vitro Drug Release Studies

Cyclosporine A, being a cyclic peptide, may be subjected to adsorption phenomena towards different materials. For this reason, before proceeding with the drug release studies, a short study to determine the behaviour of CyA towards the used materials was carried out. To prevent any CyA absorption on both dialysis membrane and glass, a Tween® 20 (0,05%) solution was used Dialysis membrane was hydrated with the Tween® 20 solution for a night, rinsed with deionized water, and then kept in a saturated water solution of CyA for 48 hours under stirring conditions. In order to quantify the drug adsorbed on the membrane, the dial-ysis membrane was rinsed, transferred into acetonitrile and kept for 24 hours under stirring conditions. The aqueous starting solution and final organic solution of CYA were analysed by HPLC analysis. The same protocol was used for the dialysis membrane hydrated with only water (control). All experiments were repeated for four times.

On the basis of the results of this experiment, the treatment of all materials used for the release studies with a Tween® 20 (0,05%) solution was recommended.

Then, 500 l of nanomicellar formulation (Nano1HAB-CyA) was transferred into a dialysis