Mechanistic Studies on Nonviral Gene Delivery to the Intestine Using in Vitro Differentiated Cell Culture Models and an in Vivo Rat Intestinal Loop
SA Cryan, CM O'Driscoll - Pharmaceutical research, 2003 - Springer
Pharmaceutical research, 2003•Springer
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 pCMV luc (firefly luciferase) plasmid, and radiolabeled …
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 pCMV luc (firefly luciferase) plasmid, and radiolabeled …
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 radiolabeled 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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