TY - JOUR
T1 - Modified pectin-based carrier for gene delivery
T2 - Cellular barriers in gene delivery course
AU - Katav, Tali
AU - Liu, Lin Shu
AU - Traitel, Tamar
AU - Goldbart, Riki
AU - Wolfson, Marina
AU - Kost, Joseph
PY - 2008/9/10
Y1 - 2008/9/10
N2 - The use of 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 pectin applicable for DNA complexation, it was modified with three different amine groups (cationic). Pectin-NH2 was prepared by modifying the galacturonic acids carboxyl groups with primary amine groups and further modified to generate pectin-T (T{double bond, short}N+H(CH3)2) and pectin-NH2-Q (Q{double bond, short}N+(CH3)3). All three 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. Pectin-NH2-Q was the most efficient carrier. Addition of chloroquine ("lysosomotropic" agent) to transfection medium substantially enhanced the HEK293 transfection, indicating that endocytosis is the preferable internalization pathway and implies on the complex inability to escape the endosome. 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. Suggesting the modified pectin is a promising non-viral carrier for targeted gene delivery to cancer cells with galactose-binding lectins on their surface.
AB - The use of 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 pectin applicable for DNA complexation, it was modified with three different amine groups (cationic). Pectin-NH2 was prepared by modifying the galacturonic acids carboxyl groups with primary amine groups and further modified to generate pectin-T (T{double bond, short}N+H(CH3)2) and pectin-NH2-Q (Q{double bond, short}N+(CH3)3). All three 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. Pectin-NH2-Q was the most efficient carrier. Addition of chloroquine ("lysosomotropic" agent) to transfection medium substantially enhanced the HEK293 transfection, indicating that endocytosis is the preferable internalization pathway and implies on the complex inability to escape the endosome. 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. Suggesting the modified pectin is a promising non-viral carrier for targeted gene delivery to cancer cells with galactose-binding lectins on their surface.
KW - Cancer
KW - Cellular barriers
KW - Non-viral gene delivery
KW - Pectin
KW - Targeted gene delivery
UR - http://www.scopus.com/inward/record.url?scp=49749087562&partnerID=8YFLogxK
U2 - 10.1016/j.jconrel.2008.06.002
DO - 10.1016/j.jconrel.2008.06.002
M3 - Article
AN - SCOPUS:49749087562
SN - 0168-3659
VL - 130
SP - 183
EP - 191
JO - Journal of Controlled Release
JF - Journal of Controlled Release
IS - 2
ER -
