|Tali, Katav - BEN-GURION UNIV.,ISRAEL|
|Marina, Wolfson - BEN-GURION UNIV.,ISRAEL|
|Joseph, Kost - BEN-GURION UNIV.,ISRAEL|
Submitted to: Controlled Release Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 2, 2008
Publication Date: June 7, 2008
Citation: Modified pectin-based carrier for gene delivery: Cellular barriers in gene delivery course. 2008. Tali Katav, LinShu Liu, Tamar Traitel, Riki Goldbart, Marina Wolfson, Joseph Kost. Journal of Controlled Release. 130:183-191. Interpretive Summary: Millions of pounds of potentially valuable pectin are contained in low-valued pulp and peel generated yearly by the U.S. sugar beet and fruit juice industries; however, less than 1% of this pectin is refined for food or other uses due to the limitation of commercial markets. Finding new uses for this biodegradable polysaccharide is critical for the long term economic viability of U.S sugar beet and citrus agribusiness. In this study, modified pectin was tested as gene carriers for use in gene therapy. The resultant pectin derivatives formed complexes with plasmid DNA and were able to deliver the DNA to host cells with high efficiency. This study demonstrated modified cationic pectin derivatives as novel gene delivery systems. The research and development of such materials will help to create new markets for pectin and eventually create new uses and value for sugar beet and citrus byproducts.
Technical Abstract: The use of biodegradable and biocompatible polysaccharides as DNA carriers has high potential for gene therapy applications. Pectin is a structural plant polysaccharide heterogeneous with respect to its chemical structure. It contains branches rich in galactose residues which serve as potential ligands for membrane receptors interaction. In order to make the anionic citrus pectin applicable for DNA complexation, pectin was modified to carry primary amine (Pectin-NH2, P-N), or quaternary ammonium groups (Pectin-N+H(CH3)2, P-Q), or a mixture of the both (P-NH2-Q). All modified pectins formed complexes with plasmid DNA as indicated by gel electrophoresis analysis. The size and morphology of pectin-NH2/DNA complexes were examined by transmission electron microscopy (TEM). Transfection experiments were carried out with human embryonic kidney cell lines (HEK293), using plasmid DNA encoding for green fluorescence protein (GFP). Transfection efficiency was analyzed by flow cytometry analysis, using FACS. P-NH2-Q was the most efficient carrier. Addition of chloroquine ("lysosomotropic" agent) to transfection medium substantially enhanced the HEK293 transfection. This indicates that endocytosis is the preferable internalization pathway. Pectin’s galactose residues contribution to transfection was examined by inhibiting pectin binding to membrane receptors (galectins), using galactose and lactose as competitive inhibitors to this interaction. Resulting reduction of transfection efficiency demonstrated the importance of pectin’s galactose residues to HEK293 transfection.