PEGylation enhances tumor targeting of plasmid DNA by an artificial cationized protein with repeated RGD sequences, Pronectin.
The objective of this study is to investigate feasibility of a non-viral gene carrier with repeated RGD sequences (Pronectin F+) in tumor targeting for gene expression. The Pronectin F+ was cationized by introducing spermine (Sm) to the hydroxyl groups to allow to polyionically complex with plasmid DNA. The cationized Pronectin F+ prepared was additionally modified with poly(ethylene glycol) (PEG) molecules which have active ester and methoxy groups at the terminal, to form various PEG-introduced cationized Pronectin F+. The cationized Pronectin F+ with or without PEGylation at different extents was mixed with a plasmid DNA of LacZ to form respective cationized Pronectin F+-plasmid DNA complexes. The plasmid DNA was electrophoretically complexed with cationized Pronectin F+ and PEG-introduced cationized Pronectin F+, irrespective of the PEGylation extent, although the higher N/P ratio of complexes was needed for complexation with the latter Pronectin F+. The molecular size and zeta potential measurements revealed that the plasmid DNA was reduced in size to about 250 nm and the charge was changed to be positive by the complexation with cationized Pronectin F+. For the complexation with PEG-introduced cationized Pronectin F+, the charge of complex became neutral being almost 0 mV with the increasing PEGylation extents, while the molecular size was similar to that of cationized Pronectin F+. When cationized Pronectin F+-plasmid DNA complexes with or without PEGylation were intravenously injected to mice carrying a subcutaneous Meth-AR-1 fibrosarcoma mass, the PEG-introduced cationized Pronectin F+-plasmid DNA complex specifically enhanced the level of gene expression in the tumor, to a significantly high extent compared with the cationized Pronectin F+-plasmid DNA complexes and free plasmid DNA. The enhanced level of gene expression depended on the percentage of PEG introduced, the N/P ratio, and the plasmid DNA dose. A fluorescent microscopic study revealed that the localization of plasmid DNA in the tumor tissue was observed only for the PEG-introduced cationized Pronectin F+-plasmid DNA complex injected. We conclude that the PEGylation of cationized Pronectin F+ is a promising way to enable the plasmid DNA to target to the tumor for gene expression.
Hosseinkhani
Hossein
H
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Tabata
Yasuhiko
Y
D000818:Animals; D004247:DNA / Q000008:administration & dosage / Q000235:genetics; D016503:Drug Delivery Systems / Q000379:methods*; D005353:Fibronectins / Q000008:administration & dosage* / Q000235:genetics; D015316:Genetic Therapy / Q000379:methods; D051379:Mice; D009842:Oligopeptides / Q000008:administration & dosage* / Q000235:genetics; D010957:Plasmids / Q000008:administration & dosage* / Q000235:genetics; D011092:Polyethylene Glycols / Q000008:administration & dosage* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000008:administration & dosage* / Q000235:genetics
D005353:Fibronectins; D009842:Oligopeptides; D011994:Recombinant Proteins; C070367:ProNectin F; D011092:Polyethylene Glycols; C047981:arginyl-glycyl-aspartic acid; D004247:DNA