A method that can deliver a drug directly to these regions or just adjacent to them and thereby localize the delivery would be more effective at targeting than the currently practiced­ strategies.

14.3  Improved Methods of Drug Delivery to the Eye Using Microneedles

A microneedle-based delivery method may be able to deliver drugs directly to intraocular tissues in a minimally invasive manner and thus provide targeted delivery. Microneedles, because of their small size, can be inserted directly into tissues of the eye and, in a variety of scenarios, target the delivery of drugs to tissues such as the corneal stroma, the sclera, and the suprachoroidal space (Fig. 14.1). We show in this chapter that microneedles, which are microscopic needles less than 1 mm long, can be used for minimally invasive intracorneal, intrascleral, and suprachoroidal delivery. We examine the capability of microneedles as a way to effectively target regions of the eye in a minimally invasive manner. As a result of their micronscale size, microneedles offer a unique way to deliver drug formulations within and between tissues of the globe. This allows the use of ocular tissues as reservoirs or conduits for drug delivery rather than just barriers to transport.

14.3.1  Intrastromal Delivery to the Cornea Using Coated Microneedles

The cornea is often treated as a barrier to drug delivery. This is especially true in glaucoma therapy using drops. The drug is initially placed on the surface of the eye but needs to be delivered to tissues further within the eye so the cornea presents itself as a barrier for this transport. However, if drugs can be delivered across the corneal epithelium and directly into the corneal stroma (i.e., intrastromal delivery), the cornea can be used advantageously for drug delivery. If drugs are deposited directly into the corneal stroma, in a minimally invasive way, the cornea can act as a reservoir to deliver drugs to the anterior segment of the eye. In cases of corneal infections or neovascularization, the cornea can be the actual target and thus intrastromal delivery can effectively target the site of disease within the cornea (Prakash et al. 2008; Tabbara and Al Balushi 2010). Furthermore, if delivery can be localized on the micron scale, then the drug can be targeted to a specific region of the cornea without exposing the whole anterior surface of the eye.

As described below, microneedles may be able to accomplish intrastromal delivery of drugs to target the cornea and anterior segment of the eye. The key advantage of using microneedles lies in the scale of the target tissue and the dimensions of the microneedle. Although corneal thickness varies, it is on the order of several hundred micrometers (Aghaian et al. 2004). A microneedle,

Fig. 14.1  Microneedles can be inserted into intraocular tissues for targeted delivery in a variety of scenarios. (a) Microneedles can be inserted into the cornea (5) for intrastromal delivery. (b) The trabecular meshwork can also be targeted by inserting a microneedle near the limbus directly into or near the trabecular meshwork. (c) Intrascleral delivery can be accomplished by inserting a microneedle into the sclera (1). (d) Microneedles inserted deeper into the sclera can target the suprachoroidal space, the region between the sclera and choroid (2), for suprachoroidal delivery. The retina (3) is located just below the choroid and the vitreous humor (4) just below that. Image of the eye was adapted from National Eye Institute, National Institutes of Health, with permission

which is on the same order of magnitude in length as the corneal thickness, can be inserted within the cornea and used to deposit the drug into the stroma. Ideally, the drug should be delivered quickly so that the microneedle can be removed within seconds from the eye. The microneedle strategy for intrastromal delivery relies on inserting the microneedle into the cornea, without penetrating across the cornea, then depositing the drug formulation within the stroma, and finally removing the microneedle as quickly as possible from the eye, thereby leaving the drug formulation behind as a depot within the cornea.

Fig. 14.2  Solid, coated microneedle. (a) A light micrograph of a single stainless steel microneedle with no coating on the surface of the microneedle. (b) A magnified view of a microneedle after coating sodium fluorescein on the surface. Sodium fluorescein is selectively coated on the microneedle and not on the base. Scale bar: 500 mm. Reproduced from Jiang et al. (2007) with permission from Association for Research in Vision and Ophthalmology

The first test of this proposed delivery method was designed to assess whether microneedles could insert into, but not across, the cornea and deposit molecules within the cornea. In order to quickly deposit the drug using a simple, inexpensive device, a solid-coated microneedle was employed. The approach is to place a dry coating of the drug on the surface of the solid microneedle, insert it into the wet interior of the cornea,­ which allows the coating to dissolve off of the microneedle, and then remove the microneedle, thereby leaving the dissolved coating within the cornea.

To test this, sodium fluorescein was used as a model compound and coated on individual solid microneedles. Figure 14.2 shows a solid stainless steel microneedle before and after coating (Jiang et al. 2007). The microneedle was tested by inserting it into the cornea of a pig eye in vitro. Figure 14.3a shows a cross-section of the cornea after insertion of a microneedle. The microneedle insertion site can be seen at the break in the corneal tissue marked by the arrow. The microneedle penetrated into the cornea without penetrating across the cornea. Figure 14.3b shows the same image under fluorescence microscopy demonstrating delivery of the fluorescein throughout the cornea. This shows that a microneedle can insert into the cornea and deliver coated compounds into the corneal stroma.

As a result of microneedle administration that bypasses the corneal epithelium and targets the corneal stroma, microneedle-based delivery should provide higher bioavailability than topical administration. To test this hypothesis, Jiang et al. studied the intraocular distribution of sodium fluorescein after intrastromal delivery using coated microneedles. The work showed that microneedles can indeed deliver molecules directly within the corneal stroma in vivo in rabbits. A single solid stainless

Fig. 14.3  A cross-section of porcine cornea after insertion of a solid stainless steel microneedle coated with sodium fluorescein. (a) A break of the corneal surface can be seen (arrow) followed by the path of the microneedle into the stroma of the cornea. The insertion site is confined to within the cornea. (b) Fluorescence micrograph of the tissue shows the sodium fluorescein (in green) has been locally delivered within the cornea near the insertion site. Scale bar: 500 mm

steel microneedle was coated with 280 ng of sodium fluorescein. The microneedle was inserted into the cornea and the concentration in the cornea, aqueous humor and lens was measured over time in vivo. As a comparison, experiments were also done applying a 3 mg dose of sodium fluorescein topically to the surface of the rabbit eye as a drop and identical measurements were made (Jiang et al. 2007).

The coated microneedle experiment showed that microneedles could be inserted into the cornea and that sodium fluorescein dissolves off the microneedle in a matter of seconds to create a depot within the stroma. In addition, sodium fluorescein levels in the anterior ocular tissues were higher than a topical application of an equivalent dose (Fig. 14.4). As an example, at the 3 h time point fluorescein concentrations in the eye were about 60 times higher than a topical application of an equivalent dose. The kinetic data also demonstrated that microneedle-based administration of fluorescein resulted in extended residence time of fluorescein as compared to topical application. In both cases, fluorescein concentrations returned to near baseline levels in the anterior segment with 24 h. The calculated bioavailability of coated sodium fluorescein delivered to the eye was 69% following microneedle administration vs. only 1% for topical administration (Jiang et al. 2007). This shows that microneedle administration effectively targeted the cornea while the topical application resulted in nearly all of the fluorescein being washed away from the eye.

These results indicate that coated microneedles should be able to deliver a therapeutically relevant molecule to the cornea and anterior segment of the eye more effectively than topical administration. Furthermore, the targeting capability of microneedles should allow for a high bioavailability of the drug and an enhanced pharmacological effect. In order to test this hypothesis, pilocarpine, a drug used to treat glaucoma, was delivered intrastromally using microneedles. Solid stainless steel microneedles were coated with approximately 1.1 mg of pilocarpine and inserted in the peripheral cornea of New Zealand white rabbits in vivo. A total of five microneedles were inserted along the circumference of the cornea targeting the

Fig. 14.4  Fluorescein concentration profiles as a function of position in the anterior chamber of the rabbit eye in vivo after administration using microneedles (a) and topical administration at 10 times the microneedle dose (b). Adapted from Jiang et al. (2007) with permission from Association for Research in Vision and Ophthalmology

peripheral cornea area. The microneedles were removed after 20 s, which was long enough to deposit the pilocarpine within the corneal stroma. Since pilocarpine causes constriction of the pupil if it reaches the ciliary muscles, the pupil size was monitored to determine if pilocarpine had reached its intended target (Jiang et al. 2007).

Fig. 14.5  Changes in rabbit pupil diameter over time in an untreated eye (open circles), in eyes treated with a topical application of 5 mg pilocarpine (gray circles), microneedles coated with 5.5 mg pilocarpine (gray square), and a topical application of 500 mg pilocarpine (black circles). Data represent the average of at least three measurements. Adapted from Jiang et al. (2007) with permission from Association for Research in Vision and Ophthalmology

Microneedle-based administration of pilocarpine caused the pupil to constrict 2.5 mm within 15 min after insertion. A similar dose of pilocarpine, 5 mg, delivered topically cause constriction of only 1 mm. In addition, the pupil began to constrict several minutes earlier when pilocarpine was administered using microneedles. This indicated that the kinetics of microneedle-based delivery of pilocarpine was faster than topical drops. When 500 mg of pilocarpine was applied topically, the pupil constricted a total of 4 mm with kinetics similar to that of 5.5 mg administered using coated microneedles (Fig. 14.5). These experiments demonstrate that a drug can be administered using microneedles and can be targeted by inserting the microneedles within the peripheral cornea. As a result, the pharmacological effect was more effective when administered through microneedles than by a less targeted approach, such as topical application (Jiang et al. 2007).

14.3.2  Intrascleral Delivery Using Coated and Hollow

Microneedles

The sclera, like the cornea, is typically seen as a barrier to transport of drugs to the back of the eye from periocular administration routes such as subconjunctival injections. Targets for posterior segment diseases such as neovascular AMD are the choroid and retina layers, which are just below the sclera. As a result, if drugs can be delivered directly to the sclera, i.e., intrasclerally, the sclera can be converted from a transport barrier to a reservoir for localized drug delivery to the underlying tissues of choroid and retina. If this can be accomplished in a minimally invasive manner, it would allow direct access to ocular tissues as natural drug delivery depots. Microneedles, given their micron dimensions, can play an important role in accomplishing this because they can specifically target the sclera and deliver drug formulations

intrasclerally. The thickness of the sclera tissue is on the order of hundreds of micrometers, which means that microneedles can be inserted intrasclerally without penetrating across the tissue and deliver a drug depot within the tissue (Olsen et al. 1998).

There are two approaches to exploit the capabilities of microneedles to deliver formulations into the sclera: solid-coated microneedles and hollow microneedles. Solid-coated microneedles can be inserted into the sclera, and the coating can dissolve off the microneedle into the sclera, after which the microneedle can be removed. This forms a local depot near the insertion site. A second approach is to use hollow microneedles to inject a formulation directly within the sclera. In this approach, a hollow microneedle is inserted into the sclera and a fluid is injected within the sclera, after which the microneedle can be removed once the desired volume is injected. A hollow microneedle functions in a way that is similar to a standard hypodermic needle, since it allows a fluid to flow through the bore of the microneedle. However, because a hollow microneedle has a microscopic orifice opening and length, a microneedle can target the sclera by spreading the fluid specifically within the sclera.

Solid-coated microneedles can allow pinpoint delivery near the insertion site within the sclera. The microneedle can be inserted into any accessible location on the sclera to deposit the coated formulation. If the drug needs to be delivered near the anterior segment of the eye, it can be inserted near the limbus. Multiple microneedles can be inserted either simultaneously as part of an array of microneedles or serially, one after the other. Figure 14.6a shows that a microneedle can penetrate into human cadaver sclera and deliver a small molecule such a sulforhodamine locally into the tissue. In addition to a small molecule, macromolecules can also be coated onto microneedles. Figure 14.6b shows the delivery of fluorescein-labeled bovine serum albumin (BSA) after intrascleral administration using a coated microneedle. The images show that microneedles can locally deliver molecules and form a depot within the sclera (Jiang et al. 2007).

It may be advantageous to not just deliver a formulation to a specific spot in the sclera, but to spread the formulation over a larger area of scleral tissue. This would allow the sclera to serve as a large reservoir for subsequent drug delivery to underlying tissues. Hollow microneedles may be able to spread a fluid within the scleral collagen matrix and accomplish intrascleral delivery of fluids. Figure 14.7 shows a hollow glass microneedle in comparison to a standard 30-gauge needle. The first reported study to show that a hollow microneedle was capable of intrascleral injection demonstrated delivery of a sulforhodamine solution within the sclera of human eyes in vitro. Bare sclera was excised from human cadaver eyes and a hollow glass microneedle was inserted into the sclera and infused with a solution. Figure 14.8 shows the delivery and spread of sulforhodamine solution within the sclera. These images show that a hollow microneedle can inject a solution intrasclerally and target the sclera tissue (Jiang et al. 2009).

An important parameter for determining effective delivery within the sclera is microneedle insertion depth. This is especially important because scleral thickness varies based on location. Scleral thickness can range from 300 mm to 1 mm within the same eye (Olsen et al. 1998). Initial experiments revealed that in addition to the

Fig. 14.6  Cross-sections of human cadaver sclera pierced using a single 750 mm-long microneedle (55° tip angle). Microneedles coated with sulforhodamine (a) and fluorescein-labeled bovine serum albumin (BSA) (b) were inserted into the sclera and deposited the coating formulation within the sclera. The arrow indicates the site of microneedle insertion. Scale bar: 250 mm. Adapted from Jiang et al. (2007) with permission from Association for Research in Vision and Ophthalmology

microneedle insertion depth, it was also important to partially retract the microneedle to flow a fluid within the sclera. The amount of fluid delivered did not vary significantly with location or the insertion depth and retraction distance. The volumes delivered were between 10 and 15 mL and all were delivered within 3 min of applied pressure. Applied infusion pressure was also varied within the different regions of the eye, and Fig. 14.9 shows the volume delivered with a constantly applied infusion pressure.

Fig. 14.7  Comparison of a hollow glass microneedle (a) to the tip of a 30-gauge hypodermic needle (b). Scale bar: 1 mm. Adapted from Patel et al. (2010) with permission from Springer Science

Fig. 14.8  Representative images of human cadaver sclera after microneedle infusion of a sulforhodamine solution. (a) Top view image of the surface of the sclera showing the infusion of sulforhodamine over an area of several square millimeters. (b) Histological section using fluorescence microscopy showing the site of microneedle insertion (arrows) and the distribution of injected sulforhodamine (in red) preferentially localized within the sclera. Adapted from Jiang et al. (2009) with permission from Springer Science

The data suggest that there is no direct correlation between applied pressure and volume delivered. As a result there may be an inherent capacity of the sclera to hold fluid and increased infusion pressure cannot overcome this limitation under the conditions tested (Jiang et al. 2009).

In addition to injecting a solution intrasclerally, injection of nanoor microparticles may be more advantageous. If designed properly, particles injected into the sclera can release a drug into the sclera tissue and provide sustained or controlled release of a drug. This can extend the residence time of the drug in the eye and reduce the administration frequency. This approach would address both of the key

Fig. 14.9  Graph showing the effect of pressure on fluid delivery into anterior, medial, and posterior regions of human cadaver sclera. Individual microneedles were inserted into the sclera with an infusion pressure of 5, 10, 15, 20, or 25 psi and the volume infused was recorded. Data are mean values (n ³ 3) with standard deviation bars. Adapted from Jiang et al. (2009) with permission from Springer Science

challenges in drug delivery to the back of the eye: targeting and extended release. Biodegradable particles can provide extended release while targeting the delivery intrasclerally by localizing the delivery near the choroid and retina tissues. Furthermore, if a single hollow microneedle can be simply inserted into the sclera and inject a particle suspension into the sclera, it may be possible to perform the procedure in a minimally invasive way.

Administration of nanoparticle suspensions using a hollow microneedle within the sclera in a minimally invasive way appears to be possible. Nanoparticles of 280 nm in diameter at concentrations up to 10 wt.% suspension were injected into the sclera with an applied pressure of 15 psi. Figure 14.10 shows the delivery of nanoparticles within the sclera tissue. Injections were performed in different regions of the sclera tissue to determine if a hollow microneedle was capable of injecting nanoparticles into all regions. Hollow microneedles were capable of injecting into all regions of the sclera, suggesting that a hollow microneedle can inject intrasclerally to any site that can be accessible on the eye. This showed that

Fig. 14.10  Representative histological images of intrascleral infusion of fluorescent nanoparticles in human cadaver sclera using a hollow microneedle as a function of nanoparticle concentration and scleral position. Microneedles were inserted into anterior, medial, and posterior regions of the sclera. A 20 mL suspension with a solids content of 0.5, 1, 5, or 10 wt.% was infused in each attempt into the tissue at a constant pressure of 15 psi. Dotted lines in each image indicate the upper and lower edges of the scleral tissue. Adapted from Jiang et al. (2009) with permission from Springer Science

nanoparticle suspensions could be injected intrasclerally similarly to the way fluids were injected into the sclera (Jiang et al. 2009).

However, administration of microparticle suspensions intrasclerally was not as straightforward. Suspensions of microparticles did not flow through the sclera ­tissue. This is in large part attributed to the spacing of the sclera collagen fibers rather than a limitation on the microneedle capability. The collagen fiber spacing is on the order of several hundred nanometers, and as a result microparticles may not easily flow within this medium (Edwards and Prausnitz 1998). To test this hypothesis, two approaches were employed to aid the movement of particles in the dense collagen matrix. One was the use of collagenase to break up the collagen structure and provide larger pathways for microparticles to flow through the sclera. The sclera tissue was either soaked in collagenase prior to injection or the collagenase was co-injected with the microparticle suspension. Both of these steps allowed infusion of the