Arto Urtti

Ocular absorption following topical delivery

Abstract Most ocular diseases are treated with topical application of eyedrops. After instillation of an eyedrop, typically less than 5% of the applied drug penetrates the cornea and reaches intraocular tissues, while a major fraction of the instilled dose is absorbed and enters the systemic circulation. Ocular absorption of topically applied ophthalmic drugs is limited by rapid precorneal drug elimination due to solution drainage and systemic absorption from the conjunctival sac. Another factor that limits ocular absorption is the corneal epithelial barrier. In the eye drugs are distributed from the aqueous humor to intraocular tissues and eliminated mainly via aqueous humor turnover and venous blood flow in the anterior uvea. Because of the publication of several reviews [1–3] on ocular drug absorption this review will focus on the most recent literature.

Efficient adventitial gene delivery to rabbit carotid artery with cationic polymer–plasmid complexes

Different lipids and cationic polymers were tested in vitro for their ability to transfect rabbit aortic smooth muscle cells and human endothelial cells with lacZ marker gene. Toxicity of the complexes was evaluated with MTT assay. Selected plasmid–polymer complexes with different charge ratios were then tested for in vivo gene transfer efficiency using adventitial gene transfer by placing a silastic gene delivery reservoir (collar) around the carotid artery. Transfection efficiency was determined by X-gal staining 3 days after the gene transfer. Based on in vitro experiments, fractured polyamidoamine dendrimers and polyethylenimines (PEI) were selected for in vivo experiments. Fractured dendrimers (generation 6, ± charge ratio of 3) had the highest in vivo gene transfer efficiency (4.4%u2009±u20091.7). PEI with mol- ecular size of 25 kDa (± charge ratio 4) was also effective (2.8%u2009±u20091.8) in this model. PEI of 800u2009kDa showed a constant but modest gene transfer efficiency (1.8%u2009±u20090.1) with all charge ratios. A low level gene transfer was also detected with naked DNA (0.5%u2009±u20090.3). No signs of inflammation were seen in any of the study groups. We show here that in vitro cell culture experiments can be used to identify efficient in vivo gene transfer methods for arterial gene therapy, but the charge ratios for each complex must be optimized in vivo. It is concluded that fractured dendrimer and PEI are efficient gene delivery vehicles and can be used for arterial gene therapy via adventitial gene delivery route.

Gene delivery and expression in human retinal pigment epithelial cells: effects of synthetic carriers, serum, extracellular matrix and viral promoters.

Abstract Non-viral gene therapy is a potential treatment to many incurable retinal diseases. To fulfill this promise, plasmid DNA must be delivered to the retinal target cells. We evaluated the efficacy of synthetic DNA complexing compounds in transfecting primary human retinal pigment epithelial (RPE) cells in vitro. Fetal human RPE cells were cultured with or without extracellular matrix (ECM), produced using calf corneal endothelial cells. Plasmids encoding nuclear localizing beta galactosidase or luciferase (pRSVLuc, pCLuc4, pSV2Luc) were complexed in water at various ± charge ratios using cationic lipids (Lipofectin, DOTAP, DOGS), polyethylene imines (25 and 750 kDa), and with degraded 6th generation starburst polyamido-amine dendrimers. Luciferase was quantified using a luminometric assay and beta galactosidase with X-gal staining. Toxicities of transfections were evaluated with the MTT-assay. Using beta galactosidase as the reporter gene naked DNA did not transfect RPE cells at measurable levels whereas 1-5% ofthecells expressed histochemically detectable amounts of the gene after transfection with cationic lipid-DNA complexes. In RPE cells, Rous sarcoma virus and cytomegalovirus (CMV) were more efficient promoters than SV40 in driving luciferase expression, and CMV was chosen for further experiments. At optimal complex charge ratios, expression levels of luciferase were > 109 light units/mg protein after transfection using dendrimers and PEI25, while transfection mediated with the other carriers resulted in luciferase expression levels of 107-109 light units/mg protein or less. In general, dendrimers and large molecular weight PEI were less toxic than cationic lipids or PEI25 to RPE cells. Serum and ECM decreased gene expression to the RPE cells with all carriers. Despite low percentage of transfected cells the transgene expression per RPE cell is high, important feature in the retinal tissue with small dimensions, in particular in the case of secreted gene products. Degraded dendrimers and high molecular weight PEI exhibited the best combination of high activity and low toxicity in RPE cell transfection.