Schmieder Anne H, Grabski Lauren E, Moore Nicole M, Dempsey Leslie A, Sakiyama-Elbert Shelly E
Department of Biomedical Engineering and Center for Materials Innovation, Washington University, St. Louis, Missouri 63130, USA.
Biotechnol Bioeng. 2007 Apr 1;96(5):967-76. doi: 10.1002/bit.21199.
The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.
本研究的目的是开发并表征一种以聚乙二醇（PEG）为主链的基因传递载体，旨在克服与当前常用的非病毒载体相关的局限性，如细胞毒性和快速清除。PEG用DNA结合肽（DBP）进行功能化，以制备能够凝聚DNA的载体（DBP-PEG）。形成质粒DNA与DBP-PEG的复合物，并通过测量粒径、zeta电位以及作为N:P电荷比（DBP-PEG氨基：DNA磷酸基团）函数的转染效率来进行表征。动态光散射表明DBP-PEG能够有效地凝聚DNA，从而产生一群粒径在250 - 300 nm范围内的颗粒。对于3.5:1及更高的电荷比，测量得到中性或略带正电的zeta电位。用编码绿色荧光蛋白（GFP）和β-半乳糖苷酶（β-Gal）基因的质粒制备的DBP-PEG/DNA复合物用于转染中国仓鼠卵巢（CHO）细胞。DBP-PEG/DNA能够转染细胞，并且在N:P比为4:1至5:1时观察到最大转染效率，对应于zeta电位为-4至+1.6 mV。测定了DBP-PEG载体对细胞活力的影响。与聚乙烯亚胺（PEI）或聚-L-赖氨酸（PLL）相比，DBP-PEG与更高比例的活细胞（约95%）相关，并且其转染效率高于PLL，但略低于PEI。这项工作的结果表明PEG可作为基因传递载体的主链。
