Sally-Ann Cryan1, Caitriona M O'Driscoll. 1. Department of Pharmaceutics and Pharmaceutical Technology, University of Dublin, Trinity College, Dublin 2, Ireland.
Abstract
PURPOSE: To identify factors influencing nonviral vector transfection in differentiated CaCo-2 and mucus-secreting coculture, CaCo-2: Ht29GlucH, cell culture models and to compare these in vitro results with in vivo transfection efficiency in rat intestine. METHODS: A range of nonviral vectors including DOTAP, Lipofectin, Superfect, PEI, and polylysine were investigated. CaCo-2 and a mucus-secreting coculture were used at 21 days. Transfection efficiency was assessed using pCMVluc (firefly luciferase) plasmid, and radio-labeled plasmid was used to determine the binding and internalization of plasmid DNA. The in vivo model used was a ligated rat intestinal loop. RESULTS: Transfection levels decreased by over 1000-fold in differentiated models relative to nondifferentiated COS-7 cells and were related to reductions in luciferase production by individual cells. Active internalization of DNA by the differentiated cells decreased. Removal of mucus by the mucolytic agent N-acetylcysteine, from the coculture system significantly reduced (p < 0.05) transfection efficiency. In vivo the transfection efficiency of PEI proved superior to DOTAP. CONCLUSIONS: Nonviral gene delivery to the hostile environment of the intestine is possible. Mechanistic studies using differentiated intestinal cell models aid identification of the rate-limiting steps to transfection and represent a more physiologically relevant approach to predict gene delivery to the intestine.
PURPOSE: To identify factors influencing nonviral vector transfection in differentiated CaCo-2 and mucus-secreting coculture, CaCo-2: Ht29GlucH, cell culture models and to compare these in vitro results with in vivo transfection efficiency in rat intestine. METHODS: A range of nonviral vectors including DOTAP, Lipofectin, Superfect, PEI, and polylysine were investigated. CaCo-2 and a mucus-secreting coculture were used at 21 days. Transfection efficiency was assessed using pCMVluc (firefly luciferase) plasmid, and radio-labeled plasmid was used to determine the binding and internalization of plasmid DNA. The in vivo model used was a ligated rat intestinal loop. RESULTS: Transfection levels decreased by over 1000-fold in differentiated models relative to nondifferentiated COS-7 cells and were related to reductions in luciferase production by individual cells. Active internalization of DNA by the differentiated cells decreased. Removal of mucus by the mucolytic agent N-acetylcysteine, from the coculture system significantly reduced (p < 0.05) transfection efficiency. In vivo the transfection efficiency of PEI proved superior to DOTAP. CONCLUSIONS: Nonviral gene delivery to the hostile environment of the intestine is possible. Mechanistic studies using differentiated intestinal cell models aid identification of the rate-limiting steps to transfection and represent a more physiologically relevant approach to predict gene delivery to the intestine.
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