Transferrin receptor (TfR) targeted therapy has emerged as a drug delivery tool which can lead to the delivery of therapeutic agents across the endothelial and epithelial barriers of choice. The potential application of the TfR as a delivery vehicle for the transport of macromolecular drugs across intestinal epithelial cells is limited by several factors, including the low level of TfR-mediated transcytosis. The GTPase inhibitor, AG10, has been shown previously to increase the apical-to-basolateral transcytosis of Tf in Caco-2 cells.
My goal has been to improve the GI absorption of the orally administered Tf-protein conjugates. For this reason the trafficking pathways of Tf was studied in order to get insight into the cellular machinery that is involved in the intracellular trafficking of Tf. Elucidating the role of the intracellular controllers of the recycling and transcytosis of Tf, such as Rab proteins, is potentially important in developing Tf as a vehicle for oral delivery of proteins.
In this thesis the intracellular trafficking of Tf through the endosomal compartments and the effect of AG10 on Tf trafficking in Caco-2 cells are described. The biochemical studies parallel with confocal fluorescence microscopy studies revealed new findings regarding the Tf trafficking pathways in Caco-2 cells. The results from these studies indicate that the Rab5-containing endosomal compartment and the Rab11-containing endosomal compartment are involved in Tf trafficking at 16°C and 37°C, respectively. The Rab5-containing endosomal compartment is localized far from the tight junction and close to the apical membrane. While the Rab11-containing endosomal compartment, which appeared as a population of cup-shaped vesicles, is localized above the tight junction. This compartment may be analogous to the apical recycling endosome (ARE) in MDCK cells. Furthermore, Rab11 is involved in the recycling of the apically endocytosed Tf in Caco-2 cells. The basolaterally endocytosed Tf does not have access to the Rab11-containing endosomal compartment. AG10 stimulates the transport of the apically and basolaterally endocytosed Tf to the Rab11-containing endosomal compartment. The existence of an internal pool for the TfR in Caco-2 cells may explain the effect of AG10 on the Tf trafficking in Caco-2 cells. AG10 may increase the Tf transport from the TfR internal pool to the late endosomal compartments involved in the transcytosis and recycling of Tf such as the Rab11-containing endosomal compartment. This could result in an enhancement of both recycling and transcytosis of Tf in Caco-2 cells. Furthermore, the ability of AG10 to inhibit the GTP-utilizing enzymes raises the possibility of its inhibitory effect on GTPase proteins such as Rab proteins. AG10 may inhibit the intrinsic GTPase activity of Rab11, resulting in a GTP-bound active form of Rab11. The presence of an active form of Rab11, which is involved in the apical recycling of Tf, may result in the enhancement of the Tf recycling in the presence of AG10.
In addition, the effect of AG10 on the Tf transport was studied. The data showed that AG10 is a selective enhancer of the TfR-mediated transcytosis in Caco-2 cells. AG10 is also a considerable enhancer of the intestinal absorption in CF-1 mice. Low potential side effects have been associated with the use of AG10 as an enhancer of Tf transport both in vitro and in vivo.
Overall, the results described in this thesis provide information regarding the intracellular processing of Tf in Caco-2 cells, data related to the effects of AG10 on the Tf intracellular processing in Caco-2 cells, data supporting a possible mechanism for the action of AG10 in Caco-2 cells, and information supporting AG10 as an enhancer of choice for the TfR-mediated transcytosis in vitro and in vivo.*
*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: QuickTime.
