Retinal Pigment Epithelium as a Barrier in Drug Permeation and as a Target of Non-Viral Gene Delivery

Retinal pigment epithelium (RPE) is a unique monolayer of cells which lie between the neural retina and the choroid. It plays an essential role in maintaining visual acuity and metabolic integrity in the retina. As a part of the blood-retina barrier, RPE restricts the molecules from blood flow and outer ocular layers from gaining access to the neural retina. Many retinal diseases may be relieved by delivery of neuroprotective or antiangiogenic factors to the retina. RPE is an interesting target for transfer of genes to induce a production of therapeutic proteins. Viral gene transfer vectors are most often used in gene therapy, but non-viral polymeric and liposomal vectors are more biocompatible and easier to produce. However, their gene transfer efficacy does not match that of viral vectors. For example, intravitreally given DNA must permeate the vitreous and the neural retina before reaching the RPE. In the present study, the limiting barriers after intravitreal non-viral gene delivery were identified. In vitro experiments with bovine vitreous and retina demonstrated that both vitreous and neural retina restrict the uptake of cationic gene complexes (DOTAP, PEI and PLL complexes) into the RPE. The large size and especially the positive charge of the complex are the reasons for a limited access into the RPE. Though, the exact mechanisms remain unclear. Oligonucleotides in solution were efficiently taken up by RPE cells, but the neural retina limits their permeation. The uptake of naked plasmid into RPE cells was very low even without the presence of vitreous or neural retina. Prolonged drug delivery to the posterior segment of the eye is a challenge in ophthalmology. After subconjunctival administration, the drug or gene product (resulting from cell or gene therapy) has to permeate across the sclera, choroid and RPE to reach the neural retina. The influence of lipophilicity (beta-blockers) and size (FITC-dextrans) of the permeants were assessed using isolated bovine choroid-RPE. For hydrophilic compounds, the choroid-RPE was 10-100 times less permeable than the sclera, whereas for lipophilic compounds the RPE and sclera were equal barriers emphasizing the important role of the RPE in permeation. The permeability of the RPE is a key factor also in the drug delivery from the systemic blood circulation into the retina. Choroid and RPE contain melanin that binds many drugs. Synthetic melanin is often used for binding studies. In the present study it was demonstrated that the melanin isolated from bovine ocular tissue and synthetic melanin differ in terms of size, surface area, shape, aggregation properties, and drug binding. Based on the data supplemented with further calculations it was estimated that the choroid-RPE contains 3-19 times more melanin bound betaxolol and metoprolol compared to the free drug. In contrast, phosphodiesterase oligonucleotides and carboxyfluorescein did not bind to melanin. In conclusion, the vitreous and neural retina are barriers to the non-viral gene transfer to the RPE. In transscleral delivery, the RPE-choroid is the rate-limiting barrier for large and hydrophilic molecules but not necessarily for lipophilic drugs. Furthermore, melanin binding modifies the pharmacokinetics of betaxolol and metoprolol at the cellular level in the posterior eye segment. National Library of Medicine Classification: QU 110, QV 132, WB 340, WW 103, WW 245, WW 250, WW 270 Medical Subject Headings: Adrenergic beta-Antagonists/ pharmacokinetics; Choroid/metabolism; Dextrans/pharmacokinetics; Drug Delivery Systems; Gene Transfer Techniques; Liposomes; Melanins; Permeability; Pigment Epithelium of Eye; Polymers; Retina; Vitreous Body