of Recent and Current Projects: (1) Sustained-Release Devices for Intraocular Drug Delivery: A number of inflammatory and neoplastic diseases of the eye are currently treated by repeated intravitreal drug injections. Transscleral delivery has emerged as a more attractive method for treating retinal disorders, because it offers localized delivery of drugs as a less invasive method compared to intravitreal administration. Numerous novel transscleral drug delivery systems, ranging from microparticles to implants, have been reported. However, transscleral delivery is currently not as clinically effective as intravitreal delivery in the treatment of retinal diseases, and this lack of success must be evaluated. Transscleral drug delivery systems require drugs to permeate through several layers of ocular tissue (sclera, choroid-Bruchs membrane, retinal pigment epithelium) to reach the neuroretina. In addition, clearance of administered drugs occurs by uptake into the conjunctival and choroidal blood and possibly into lymph. This project involves the development of a method to obtain sustained release of a protein molecule (ovalbumin) to posterior eye tissues. We have investigated the use of such sustained drug release devices that could release drugs into the body for periods as long as months. We tested the use of a thermosetting polymer, ReGel (Protherics, a BTG PLC company.), as an injectable agent for long term release of drugs into the eye. This technique would eliminate the need for frequent invasive bolus liquid injections. Our experiments used subconjunctival injections of ReGel containing fluorescently labeled ovalbumin into rat eyes. In the rat experiments, a drug marker, in the form of fluorescently-labeled ovalbumin (Alexafluor-ova), was incorporated into the ReGel solution, which is liquid at room temperature. The ReGel was then injected into the subconjunctival space of rats, where it formed a slow releasing gel at 37 centigrade. Animals were sacrificed at serial time points from one hour up to 14 days and the enucleated eyes were examined in histological sections by fluorescent microscopy and by tissue extraction for the quantitative assessment of fluorescently labeled protein. Our data indicates that we can obtain measurable levels of intact Alexa-ova for up to 14 days in sclera, choroid, and retina tissues using the ReGel system. (2) Influence of Proteoglycans on Axonal Growth: Chondroitin sulfate proteoglycans (CSPGs) are major components of glial scars that form soon after spinal chord injury. It is believed that CSPGs significantly inhibit axonal growth and regeneration. CSPGs consist of a protein core with large glycosaminoglycan (GAG) sugar side chains. Removal of the GAG chains by chondroitinases has been shown to restore neuronal growth activity. We are seeking to determine which of the specific GAG sugars, chondroitin sulfate A or chondroitin sulfate C, is responsible for the neuronal inhibition. We are also planning to study potential sustained release devices that can release the chondroitinases into the injury sites to reduce the presence of the GAG sugars on the proteoglycans and thus encourage axonal regeneration. Our initial experiments studied the effects of whole CSPGs on neuronal growth. We determined the release and distribution characteristics of nanoparticle-encapsulated CSPGs through a collagen test matrix. The effect of the CSPGs on the sprouting of PC-12 cells which were imbedded in the collagen matrix was measured. Cell growth and sprouting was monitored by confocal microscopy. We found a dose response in which cell growth was inhibited more at radial distances closer to the nanoparticle injection site where CSPG concentration would be the highest. Another phase of the project tested the ability of a sustained release polymer, ReGel, a thermosetting gel to release CSPGs into a collagen test matrix where neuronal cells will grow. Future phases of this work propose a series of in vitro experiments to study the effects of the specific CSPG sugars on the growth and sprouting of neuronal PC-12 cells into a collagen matrix. Cell growth will be monitored by microscopy. In addition, we will test the release and distribution of chondroitinase enzyme which will be delivered from nanoparticles or ReGel into the center of an in vitro gel matrix. The effectiveness of this delivery method on cleaving the inhibiting sugar moieties will be studied by monitoring of the inhibition of neuronal cell growth with confocal microscopy. Subsequent in vivo experiments would investigate the effectiveness of a nanoparticle or ReGel enzyme delivery system on enhancing nerve regeneration by administering enzyme injections into rat spinal chord after chord injury or in injured goldfish optic nerve, another model of nerve regeneration.
