Mechanistic studies on nonviral gene delivery to the intestine using in vitro differentiated cell culture models and an in vivo rat intestinal loop.
OBJECTIVE
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.
Cryan
Sally-Ann
SA
Department of Pharmaceutics and Pharmaceutical Technology, University of Dublin, Trinity College, Dublin 2, Ireland.
O'Driscoll
Caitriona M
CM
D000818:Animals; D019556:COS Cells; D018938:Caco-2 Cells / Q000378:metabolism; D018929:Cell Culture Techniques; D004337:Drug Carriers; D005822:Genetic Vectors; D019073:HT29 Cells / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D008055:Lipids; D008297:Male; D023421:Models, Animal; D009093:Mucus / Q000378:metabolism; D010455:Peptides; D011108:Polymers; D051381:Rats; D017208:Rats, Wistar; D014162:Transfection
D004337:Drug Carriers; D008055:Lipids; D010455:Peptides; D011108:Polymers