Hosseinkhani Hossein, Tabata Yasuhiko
Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
J Control Release. 2004 May 31;97(1):157-71. doi: 10.1016/j.jconrel.2004.02.025.
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.
本研究的目的是探讨具有重复RGD序列的非病毒基因载体（Pronectin F+）在肿瘤靶向基因表达中的可行性。通过将精胺（Sm）引入羟基对Pronectin F+进行阳离子化，使其能够与质粒DNA形成聚离子复合物。对制备的阳离子化Pronectin F+进一步用末端具有活性酯和甲氧基的聚乙二醇（PEG）分子进行修饰，形成各种引入PEG的阳离子化Pronectin F+。将不同程度PEG化或未PEG化的阳离子化Pronectin F+与LacZ质粒DNA混合，形成各自的阳离子化Pronectin F+-质粒DNA复合物。质粒DNA与阳离子化Pronectin F+和引入PEG的阳离子化Pronectin F+通过电泳形成复合物，与PEG化程度无关，尽管与后一种Pronectin F+形成复合物需要更高的N/P比。分子大小和zeta电位测量结果显示，与阳离子化Pronectin F+形成复合物后，质粒DNA的大小减小到约250 nm，电荷变为正值。对于与引入PEG的阳离子化Pronectin F+形成的复合物，随着PEG化程度的增加，复合物的电荷变为中性，几乎为0 mV，而分子大小与阳离子化Pronectin F+相似。当将PEG化或未PEG化的阳离子化Pronectin F+-质粒DNA复合物静脉注射到携带皮下Meth-AR-1纤维肉瘤肿块的小鼠体内时，引入PEG的阳离子化Pronectin F+-质粒DNA复合物特异性地提高了肿瘤中的基因表达水平，与阳离子化Pronectin F+-质粒DNA复合物和游离质粒DNA相比，提高程度显著。基因表达增强水平取决于引入PEG的百分比、N/P比和质粒DNA剂量。荧光显微镜研究显示，仅在注射了引入PEG的阳离子化Pronectin F+-质粒DNA复合物后，才观察到肿瘤组织中质粒DNA的定位。我们得出结论，阳离子化Pronectin F+的PEG化是使质粒DNA靶向肿瘤进行基因表达的一种有前景的方法。
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