Kneuer C, Sameti M, Bakowsky U, Schiestel T, Schirra H, Schmidt H, Lehr C M
Department of Biopharmaceutics and Pharmaceutical Technology, Saarland University, 66041 Saarbrücken, Germany.
Bioconjug Chem. 2000 Nov-Dec;11(6):926-32. doi: 10.1021/bc0000637.
Diverse polycationic polymers have been used as nonviral transfection agents. Here we report the ability of colloidal silica particles with covalently attached cationic surface modifications to transfect plasmid DNA in vitro and make an attempt to describe the structure of the resulting transfection complexes. In analogy to the terms lipoplex and polyplex, we propose to describe the nanoparticle-DNA complexes by the term "nanoplex". Three batches, Si10E, Si100E, and Si26H, sized between 10 and 100 nm and with zeta potentials ranging from +7 to +31 mV at pH 7.4 were evaluated. The galactosidase expression plasmid DNA pCMVbeta was immobilized on the particle surface and efficiently transfected Cos-1 cells. The transfection activity was accompanied by very low cytotoxicity, with LD(50) values in the milligrams per milliliter range. The most active batch, Si26H, was produced by modification of commercially available silica particles with N-(6-aminohexyl)-3-aminopropyltrimethoxysilane, yielding spherical nanoparticles with a mean diameter of 26 nm and a zeta potential of +31 mV at pH 7.4. Complexes of Si26H and pCMVbeta plasmid DNA formed at w/w ratios of 10 were most effective in promoting transfection of Cos-1 cells in the absence of serum. At this ratio, >90% of the DNA was associated with the particles, yielding nanoplexes with a net negative surface charge. When the transfection medium was supplemented with 10% serum, maximum gene expression was observed at a w/w ratio of 30, at which the resulting particle-DNA complexes possessed a positive surface charge. Transfection was strongly increased in the presence of 100 microM chloroquine in the incubation medium and reached approximately 30% of the efficiency of a 60 kDa polyethylenimine. In contrast to polyethylenimine, no toxicity was observed at the concentrations required. Atomic force microscopy of Si26H-DNA complexes revealed a spaghetti-meatball-like structure. The surface of complexes prepared at a w/w ratio of 30 was dominated by particles half-spheres. Complex sizes correlated well with those determined previously by dynamic light scattering.
多种聚阳离子聚合物已被用作非病毒转染剂。在此，我们报告了具有共价连接阳离子表面修饰的胶体二氧化硅颗粒在体外转染质粒DNA的能力，并尝试描述所得转染复合物的结构。类似于脂质体和多聚体的术语，我们建议用“纳米体”一词来描述纳米颗粒 - DNA复合物。评估了三批粒径在10至100nm之间、在pH 7.4时zeta电位为+7至+31mV的Si10E、Si100E和Si26H。半乳糖苷酶表达质粒DNA pCMVbeta固定在颗粒表面，并有效转染Cos - 1细胞。转染活性伴随着非常低的细胞毒性，半数致死剂量（LD50）值在毫克每毫升范围内。活性最高的批次Si26H是通过用N -（6 - 氨基己基）- 3 - 氨丙基三甲氧基硅烷修饰市售二氧化硅颗粒制备的，得到平均直径为26nm、在pH 7.4时zeta电位为+31mV的球形纳米颗粒。Si26H与pCMVbeta质粒DNA以10的重量比形成的复合物在无血清条件下促进Cos - 1细胞转染最为有效。在此比例下，>90%的DNA与颗粒结合，产生表面带净负电荷的纳米体。当转染培养基补充10%血清时，在30的重量比下观察到最大基因表达，此时所得颗粒 - DNA复合物具有正表面电荷。在孵育培养基中存在100μM氯喹时，转染显著增加，达到约60kDa聚乙烯亚胺效率的30%。与聚乙烯亚胺不同，在所需浓度下未观察到毒性。Si26H - DNA复合物的原子力显微镜显示出类似意大利面肉丸的结构。以30的重量比制备的复合物表面以颗粒半球为主。复合物大小与先前通过动态光散射测定的结果相关性良好。
