Keywords

This chapter briefly revisits the corneal anatomy and physiology to better understand the barriers that pharmacologic agents have to overcome in order to reach a therapeutic threshold. Furthermore, we discuss several common corneal pathologic processes and their current pharmacologic treatments. Finally, we touch upon current and future applications of nanotherapeutics and ophthalmic drug delivery in the treatment of corneal disease.

25.1 Anatomy

The cornea of the eye is the anterior most avascular clear tissue segment of the eye, which functions predominantly as the main refractive medium of the eye (not the lens, contrary to popular belief). It also serves as a protective barrier against ocular penetration from pathogenic microbes, foreign bodies, and pharmacologic agents. Familiarity of the corneal anatomy is important to understand how the cornea maintains clarity and also how nanotherapeutic agents can overcome structural barriers to treat pathological corneal conditions.

There are five distinct corneal layers. These include (from anterior to posterior): the corneal epithelium, Bowman’s layer, corneal stroma, Descemet’s layer, and the corneal endothelium. Each is unique in structure and function and serves an important role in maintaining corneal integrity .

The corneal epithelium is composed of stratified squamous cells and reflects the anterior most cellular surface of the cornea. It measures approximately 50 μm in width ([21], 197). This layer is further segregated into the basal most columnar cells, slightly anterior wing cells, and surface cells. The surface cells, at their apical edge contain microvilli that intimately interact with the tear film to provide a lubricating medium. These same surface cells and their microvilli are also involved in increasing contact time between pharmacologic ophthalmic agents and corneal epithelial cells. Furthermore, the desmosome helps form tight bonds or adhesions between adjacent surface cells and helps to form an impermeable barrier against bacterial entry. Epithelial cells are shed routinely and regenerate from corneal limbal stem cells. Basal cells are fixed to the epithelium basement membrane through hemi-desmosomes. The corneal epithelium, through its tight junctions, is a lipophilic agent.

Together, the corneal epithelium basal cells and the stromal keratocytes help produce an acellular basement membrane-like layer of collagen fibrils termed the Bowman’s layer. This layer does not regenerate if destroyed and, as a result, may cause corneal edema and scarring if damaged.

The corneal stroma is the thickest layer, measuring approximately 500 μm and composed of keratocytes, type I collagen, and glycosaminoglycans (specifically keratin sulfate) [22]. The cornea maintains its clarity because stromal proteins and cells are arranged in a parallel plane helping preserve an optically clear corneal media. The regular arrangement of the corneal stroma also allows for easy dissection during lamellar surgery. In contrast to the corneal epithelium, the stroma is a hydrophilic medium.

Descemet’s membrane is a true basement membrane produced by the corneal endothelium and measures approximately 10 μm [22]. Descemet’s membrane is composed of type IV collagen fibrils and, unlike Bowman’s layer, may be synthesized if damaged. It serves to anchor the underlying endothelial cells to the corneal stroma.

The corneal endothelium is the posterior most cellular layer of the cornea, separating the corneal stroma from the anterior chamber. More importantly, it serves an important role in maintaining corneal clarity through energy-dependent channel proteins that help keep the cornea dehydrated. Composed of a single layer of hexagonal cells, the corneal endothelial cell density is fixed from birth and, if damaged, does not regenerate. Fuchs’ dystrophy is a pathological corneal condition associated with the loss of corneal endothelial cells. If enough endothelial cells are damaged, the cornea begins to swell, and edema ensues.

It is important to note that the cornea itself is completely devoid of any vascular supply and receives oxygen through the air/tear film complex and glucose from the aqueous fluid. In fact, sub-threshold therapeutic concentrations of topical ophthalmic medications are partly a result of lymphatic and vascular flow of pre-corneal space. By the time ophthalmic drugs are delivered to the cornea, therapeutic concentrations have decreased substantially. Other limitations to drug delivery to the front of the eye include unintended dilution of medications through contact with the tear film and drainage through the nasolacrimal system [1]. The natural blinking phenomenon and normal tear drainage in patients greatly reduce the total concentration of medication which eventually reaches the pre-corneal space.

Once it reaches the cornea, there are static structural barriers that must be overcome including tight junctions between corneal epithelial cells and inherent molecular and electrical charges of the epithelium and the stromal tissue [20].

25.2 Current Modes of Ophthalmic Drug Delivery

In general, there are three main mechanisms of ophthalmic drug delivery: solutions, suspensions, and ointments. We discuss benefits and disadvantages of each mode of delivery in the following section.

Ophthalmic topical solutions have distinct benefits over other systemic formulations (intravenous, oral, and even intravitreal) but also have some major disadvantages. The benefits lie in the fact that topical medications, in general, are poorly absorbed systemically and, as a result, systemic toxicity, such as renal failure or hepatic dysfunction is less likely. At the same time, if a topical preparation of a drug does reach the systemic circulation, the likelihood of an adverse pharmacologic event may be higher because it bypasses first-pass metabolism from the liver. As mentioned previously, ophthalmic solutions as a means of drug delivery are not an ideal mechanism because of its poor bioavailability, minimal corneal contact time, and dependence on preservatives.

Ophthalmic ointments are another means of ophthalmic drug delivery, which in itself have certain benefits over ophthalmic solutions. For instance, because ointments, by definition, have a higher viscosity than their solution counterparts, they promote longer surface contact with the cornea and allow for an improved therapeutic effect. This benefit is even more significant in pediatric populations where sufficient topical drop administration is limited by patient cooperation and excessive tearing. A major disadvantage to ophthalmic ointments is that it limits accessibility of other ophthalmic medications to the ocular surface. Additionally, ointments cause blurry vision and are not recommended for daytime use [3].

25.3 Future Ophthalmic Drug Delivery Methods: Nanotherapeutics

Recent focus on ophthalmic drug delivery has shifted to include the use of nanotherapeutics to improve therapeutic concentrations, corneal penetration, and reduce toxicity. Nanotherapeutics refers to the use of nanoparticles or micromolecules, ranging from 1 to 100 nm in size, that are genetically or pharmacologically manufactured to provide a means of drug delivery at the cellular level. There are several subgroups of nanoparticles that have been implicated for its use specifically with corneal pathology [2].

The use of nanotherapeutics for ophthalmic drug delivery has become a very hot topic of interest in recent years as physicians, scientists, and pharmacists find ways to overcome static and dynamic barriers to ophthalmic delivery. Current modalities of delivery include solutions, suspensions, and ointments. While each has its own advantages and disadvantages, overall, their effectiveness is limited by the tear film, the nasolacrimal drainage system, the blink reflex, the scleral/conjunctival vascular and lymphatic flow, and the tight junctions between corneal epithelial cells. The majority of nanotherapeutic research involves local therapy against macular degeneration, retinitis pigmentosa, and glaucoma management and treatments. The following sections describe recent nanotherapeutic developments in relationship to corneal pathology.

25.4 Colloids

Recent progress has been made in identifying unique modalities for ophthalmic drug delivery, specifically in the scope of corneal pathology. Current advances include reliance on colloids through delivery of medications via liposomes, microemulsions, and nanoemulsions. Colloids are small solid particles that do not dissolve in a solution and, unlike other solid particles, have an ability to scatter light [11].

25.5 Liposomes

Liposomes are microvesicles made of a surrounding lipid bilayer coat and a central aqueous center. The aqueous center allows for incorporation of water-soluble drugs, while lipid soluble drugs can be attached to the lipid coating bilayer membrane [13]. This amphiphilic property of liposomes makes them an ideal drug delivery mechanism because it improves corneal penetration through the hydrophobic epithelium and the lipophilic corneal stroma. Additionally, due to the inherent properties of liposomes, they provide a longer half-life and a persistent steady release of medication than topical applications of the same medication. In fact, liposomes have been manipulated to include vitamins, specifically vitamin A and C, to promote corneal hydration in the treatment of dry eye syndrome and also in the delivery of idoxuridine to treat herpetic keratitis to prevent epithelial toxicity [4].

Unlike ophthalmic suspensions and solutions, liposomes and microemulsions provide a means for greater bioavailability, sustained and controlled release of medications, and allow for bypass of the blood ocular barriers. By exchanging different phospholipids on the cell membrane, physician-scientists are able to selectively modify and assemble colloids which would target specific cells. It is important to note that liposomes may be manipulated to overcome the net negative charge of the corneal epithelium. Catatonic liposomes have better permeability and penetration into the corneal stroma, a characteristic which is useful when targeting medications to the stroma [5]. Unfortunately, liposomes are easily biodegraded and are relatively unstable. Historical studies centered on liposomal carrier systems, specifically penicillin G and indoxole in rat corneas, have established liposomes as adequate alternative carrier mechanism with impressive corneal and aqueous penetration and permeability [19].

In contrast, niosomes are a cheaper and more stable alternative to liposomes because they are made up of nonionic surfactant vesicles yet maintain a bilayer amphiphilic structure. Their lipid surfactant membrane and aqueous center make niosomes biodegradable, stable, and flexible, while reducing toxicity and cost. Furthermore, the nonionic surfactant makes niosomes less likely to promote an inflammatory reaction. Current studies in the use of niosomes revolve around the delivery of ophthalmic mydriatics including cyclopentolate and anti-ocular-hypertensive agents such as timolol. These studies show promising results in in vivo animal models by establishing increased intracameral concentrations than their solution and suspension counterparts [15].

25.6 Microemulsions

In contrast, microemulsions are nanoparticles made up of two immiscible liquid moeities: usually, a hydrophilic agent such as water and a hydrophobic oil with adjunct surfactant. Microemulsions are easily created in the lab, requiring little to no energy expenditure. The amphiphilic property of microemulsions, in addition to their small size, allow for easy penetration, accessibility, and prolonged release of therapeutic drugs to the target tissue through the formation of intracorneal depots. Microemulsions exist through the formation of micelles [14]. Current experimental therapeutic microemulsions that have been developed incorporate anti-inflammatory nonsteroidal agents, steroids, and ocular anti-hypertensives to treat difficult ophthalmic disease entities. Their benefits reside in the fact that they are not subject to pre-corneal barriers of the conjunctival lymphatic and vascular system and are able to circumvent structural impediments of the cornea [6].

The creation and use of voriconazole microemulsions in the treatment of fungal corneal ulcers has been researched, and early studies ex vivo show a clear increase in voriconazole concentrations after 12 h in the corneal tissue when compared to suspensions. In vivo studies are being planned [7].

Similarly, dry eye syndrome is another corneal pathological entity which has benefited from the use of nanotherapeutics for treatment. Colloidal application of topical cyclosporine A, through the creation of catatonic micelles, has been widely successful in treating patients with refractory dry eyes and has resulted in improved symptoms, patient cooperation, and improved bioavailability, more so than their oil-based microemulsion counterparts [8].

25.7 Transporter Proteins

Another pattern of how nanotherapeutics is shaping the way corneal disease is managed is through the advent of transporter proteins. These membrane transport proteins can selectively target and bind ligands and allow for passage into and out of the cells. Recent advances to transporter proteins have enabled physician-scientists to manipulate this system so that certain drug moieties may be combined to the ligand to allow intracellular diffusion of the medication [16]. There are two specific types of transporter proteins: efflux and influx proteins. Efflux transport proteins are energy-dependent proteins that enable for targeted ligands to be removed extracellularly. In contrast, influx transport proteins rely on a solute transport mechanism down concentration gradients without requiring energy. Transporter proteins allow for selective drug delivery of medications to the epithelial of the cornea, conjunctiva, and sclera.

These transport proteins have been vital in the delivery of antiviral agents for the treatment of herpetic epithelial/stromal keratopathy. By binding pro-drugs of acyclovir and ganciclovir to certain ligands, scientists have been able to manipulate transport proteins to allow for influx and efflux of these agents and increase the intracellular concentration of these antivirals (ocular drug delivery). Furthermore, transport proteins are being tested in animal models for adjunct vitamin C delivery to the cornea [16].

Efflux transport proteins appear to serve as an inherent protective mechanism to the cell by ridding itself of molecules that the cell believes are toxic [9]. Future research may evolve into selectively targeting these genes in hopes to increase intracellular concentrations of certain medications by limiting its exit out of the cell [16].

25.8 Dendrimers

Dendrimers are polymers of repetitive branching molecules that form structurally symmetric spherical nanomolecules in a concentric fashion, likewise to the layers of an onion. Each tier of repetitive monomeric subgroups encompasses a core, and it is through alterations of the functional subgroups that allow specific interactions between different drugs, target cells, and pathogens [4]. Dendrimers essentially serve as transporters for nanoparticles, by either incorporating the target drug in the core or by conjugating it through one of its subgroups. Current use of dendrimers in ophthalmic delivery has only been shown to be effective in gene transplant, and through instillation of ophthalmic miotic and mydriatic agents.

25.9 Ocular Iontophoresis

Ocular iontophoresis involves using an electrical stimulus to drive pharmacologic agents through the tissue planes by taking advantage of the fact that similarly charged agents repel each other and opposite charged agents attract. This system enables scientists and ophthalmologists to manipulate medications by introducing a net charge, and then, applying an electrical stimulus so that the medication can be pushed or pulled through a transcorneal approach. Furthermore, net migration of medications is also assisted through electroosmosis, which relies on particles moving with a current from the anode to the cathode [10].

Introduction of antibiotics, antivirals, antifungals, and anti-inflammatory medications through the use of ocular iontophoresis has proved to be successful in the experimental stage. Every class of antibiotics has been studied and found to successfully penetrate the cornea for the treatment of bacterial keratitis. In fact, recent advances of ocular iontophoresis have introduced anesthetics such as lidocaine and epinephrine as viable medications that can successfully be concentrated into the eyelids for oculoplastic procedures. The use of an electric probe to electrically stimulate the transportation of medications through the corneal planes appears to be a safe, painless, effective, and fast alternative to current invasive measures [11].

The idea of reverse iontophoresis is another up-and-coming nanotherapeutic procedure, which serves to produce a unique avenue for ophthalmic drug delivery. In vitro studies with hemosiderin-stained corneas illustrate the successful extraction of hemosiderin deposits from the corneal stroma and endothelium. By altering the net charge of iron, an electrical stimulus was used to remove the residual hemosiderin granules from the corneal tissue [17]. This is a particularly useful strategy in the treatment of complicated hyphema after trauma with resulting endothelium blood staining. Current treatments consist of emergent anterior chamber washout and, if staining is severe, corneal transplantation.

The use of the above nanoparticles, specifically liposomes and microemulsions, can be concurrently combined with the current uses of contact lenses to provide a unique combination of ophthalmic drug delivery. Current contact lenses are composed of a polymeric material that mimics the ocular surface shape and comfortably rests on the ocular surface. While pre-soaking contact lenses with a drug may initially prolong the duration of a therapeutic effect, contact lenses are still subject to the same limitations as ophthalmic solutions, specifically, the amount of drug that can be loaded to the contact lenses and a half-life only of hours. Soaking a pair of contact lenses after it has been placed on the eye does not fare much better. However, combining nanoparticles, such as microemulsions and liposomes onto contact lenses, not only provides an immediate release of the drug lasting several hours, but also provides a sustained release for several days by taking advantage of the hydrophobic and hydrophilic properties of oil and water emulsions. Drugs mixed with aqueous agents tends to diffuse immediately, while drugs soaked in oil takes days to diffuse [4].

25.10 Sonophoresis

Ultrasound has been used for generations in the evaluation and diagnosis of ophthalmic pathology when the visualization of the posterior pole is difficult or obstructed as in the case of an evaluation of the retina during vitreous hemorrhage or mature cataracts. Recent advances have enabled scientists to link high-frequency sound waves to ophthalmic medications in efforts to facilitate drug delivery into the cornea and even the retina. Sonophoresis or ultrasound-mediated delivery of ophthalmic medications relies on the creation of cavitation or gas bubbles that can penetrate through intact ocular tissue. The use of an ultrasound, often at a frequency of greater than 800 kHz in conjunction with a coupling medium (often the medication to be infused), creates micro-bubbles which oscillate to form pores that enable cellular and vascular permeability. The use of sonophoresis in rabbit models to deliver ophthalmic dexamethasone was studied and found that ultrasound frequency at 800 kHz enhanced and optimized corneal drug delivery. However, limitations to the widespread use of sonophoresis stem from the concerns of thermal and mechanical damage to the avascular cornea, lens, and vitreous from the high-frequency and prolonged ultrasound energy [18].

25.11 Microneedles

Microneedle therapy will be briefly introduced as a potential new ophthalmic drug delivery option in the treatment of anterior segment and corneal disease processes.

The use of microneedles as a therapeutic means has gained traction in recent years. Microneedle therapy has been used for some time in the delivery of vaccines and medications intradermally, with overall good effect. However, recent use of microneedles, which are customized needles, ranging in size from 25 to 2000 μm in length, provides a means of localized delivery without causing pain and extensive tissue damage. While microneedles are very effective in the treatment of posterior segment disease, there are novel applications for their use in corneal pathology. Current innovation for microneedle therapy is used for the treatment of cystoid macular edema and posterior uveitis via intrascleral injection of steroids directly into the suprachoroidal space. This provides faster entry and higher concentrations in the retinal space [12]. Current corneal applications of microneedle therapy are limited to the introduction of gene vectors into the epithelial and stromal cells. Further studies which link microneedles with coated antibiotics for the treatment of infectious keratitis are underway; however, extensive corneal damage from the underlying infection makes microneedles difficult to use.