Corneal Epithelial Cell Culture Model for Pharmaceutical Studies

In general, drug absorption into the eye from eye drops is limited. Only 1-7% of the dose eventually reaches aqueous humor since corneal epithelium effectively limits drug delivery into the eye. Gene therapy offers new therapeutic possibilities in ophthalmology, but delivery is an important issue in this case. Ocular drug delivery experiments require sacrification of at least five animals at each time point in the drug concentration profile. Improved corneal cell culture model would therefore be useful in ocular drug and gene delivery experiments, and might reduce the need for animal experiments. The aim of the study was to develop a cell culture model of corneal epithelium for pharmaceutical studies. The cell culture model was tested as a tool in drug and gene delivery experiments. Immortalised human corneal epithelial cells (HCE) were grown on collagen or laminin covered permeable support filters with or without feeder fibroblasts. After air-lift the cells stratify and differentiate forming epithelium approximately seven cell layers thick with flattened superficial cells, tight junctions and microvilli. In the optimised cell model the penetration of blockers increased with lipophilicity following an almost similar sigmoidal relationship with that of excised rabbit cornea. Paracellular permeability in the HCE-model was generally found to be slightly higher than in the excised rabbit cornea. The HCE-model has larger paracellular pores at lower density than the excised cornea, but overall paracellular space was fairly similar. The HCE-model has esterase activity. Rabbit corneal epithelium in vivo was transfected using non-viral liposomes (1,2-dioleoyl-3trimethylammonium-propane; DOTAP and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine DOPE) to secrete transgene product (SEAP; a secreted form of human placental alkaline phosphatase) into the tear fluid and aqueous humor. Furthermore, suitability of the differentiated corneal epithelial cell culture for transfection studies was evaluated with DOTAP/DOPE and cationic polymer (polyethyleneimine; PEI). The transfection levels decreased with the increased differentiation of HCE cells. PEI was particularly effective in transfecting the dividing cells but ineffective in the differentiated cells. DOTAP/DOPE also showed high activity in differentiated cell cultures. Significant SEAP expression was seen for at least three days after in vivo transfection in the tear fluid and aqueous humor. Rates of SEAP secretion to the basolateral side of differentiated HCE-cells and into the aqueous humor in vivo were in the same range showing the predictive applicability of the cell model. In conclusion, the morphology, physical barrier and permeability properties demonstrate that the HCE-model closely resembles those of the excised rabbit cornea. Corneal epithelium can be transfected topically to reach prolonged protein secretion into the tear fluid and aqueous humor, and the levels of this protein secretion can be predicted correctly with the cell culture model. National Library of Medicine Classification: WW 166, WW 220, WB 340, QZ 52 Medical Subject Headings: Eye Diseases/drug therapy; Drug Administration Routes; Drug Delivery Systems; Gene Transfer Techniques; Transfection; Liposomes; Ophthalmic Solutions; Cornea; Epithelium, Corneal; Cells, Cultured; Permeability; Esterases; Alkaline Phosphatase; Rabbits ACKNOWLEDGEMENTS The present work was carried out in the Department of Pharmaceutics, University of Kuopio, during the years 1997-2006. I owe my deepest gratitude to my principal supervisor Professor Arto Urtti. His continuous optimistic attitude, support and patience have guided me throughout this project. I am greatly indebted to my other supervisors Pekka Suhonen, Ph.D., and Docent Raija Tammi, M.D., Ph.D. for their guidance and valuable comments during these years. Professor Hanna Tahti and Docent Juha Holopainen, M.D., Ph.D. are gratefully acknowledged for reviewing the manuscript, their constructive criticism and suggestions for improvement. My warm thanks go to Patrick Kehoe, Ph.D. from the University of Bristol, U.K, for revising the language of this dissertation. I am also honoured that Professor Jouni Hirvonen accepted the invitation to act as my opponent. I wish to thank Professor Jukka Monkkonen, Dean of the Faculty of Pharmacy, and Professor Kristiina Jarvinen, Head of the Department of Pharmaceutics, for providing me with excellent working facilities. I am deeply grateful to my co-authors Veli-Pekka Ranta, Ph.D., Anu Talvitie, M.Sc., Kati-Sisko Vellonen, M.Sc., Joni Palmgren, Ph.D., Mirka Laavola, M.Sc., Kaisa Mari Hamalainen, Ph.D., Docent Seppo Auriola, Ph.D., Margit Hornof, Ph.D., Docent Kai Kaarniranta, M.D., Ph.D., and Pinja Johansson, M.Sc. for their remarkable contribution to this work. My sincere thanks go to my collaborators Paula Saarinen-Savolainen, Ph.D., Marjukka Suhonen, Ph.D., Mrs. Lea Pirskanen, Seppo Ronkko, Ph.D., Docent Matti Elomaa, Ph.D., Mika Reinisalo, M.Sc., and Professor Paavo Honkakoski for their help in many different stages of this work. I also owe so much to Marika Ruponen, Ph.D., Eliisa Mannermaa, M.D., and Ms. Marja Lappalainen regarding scientific and not so scientific matters. My special thanks go to the present and former staff of the Department of Pharmaceutics and shared staff of the Faculty of Pharmacy. It has been good to work with you during these years. I also thank the Department of Pathology and Forensic Medicine, Laboratory of Electron Microscopy and National Laboratory Animal Center for skilful technical assistance. I express my kindest thanks to my close friends and relatives. I am so lucky to have such wonderful people in my life! My dearest thanks I owe to my loving family. Your never-ending love, care and support have given strength to me every day. I dedicate this thesis to all of you. The Academy of Finland, Emil Aaltonen Foundation, the Finnish Cultural Foundation, the Finnish Cultural Foundation of Northern Savo, Graduate School of ESPOM, Julia von Wendt Foundation and the Research and Science Foundation of Farmos (Finland) are acknowledged for the financial support.