Hosseinkhani Hossein, Aoyama Ternyoshi, Yamamoto Shingo, Ogawa Osamu, Tabata Yasuhiko
Institute for Frontier Medical Sciences, Kyoto University, Japan.
Pharm Res. 2002 Oct;19(10):1471-9. doi: 10.1023/a:1020400514990.
The purpose of this study is to examine the ultrasound (US)-enhanced gene expression by the complexes of a plasmid DNA with gelatin derivatives of aminization.
Gelatin derivatives with different introduced extents of ethylenediamine (Ed), spermidine (Sd), and spermine (Sm) were prepared with a water-soluble carbodiimide. The molecular size and zeta potential of the gelatin derivatives before and after complexation with the plasmid DNA were examined. After incubation with the complexes with or without US exposure, the DNA expression of rat gastric mucosal cells was measured to evaluate the effect of the type of gelatin derivatives on their gene expression. The cell uptake of the complexes, the cell viability, and the buffering effect of gelatin derivatives were examined.
The apparent molecular size and zeta potential of gelatin derivatives became larger as their aminization extent increased although the Sm gelatin derivative of higher aminization showed a larger value than other corresponding derivatives. Irrespective of the type of gelatin derivatives, the apparent molecular size of plasmid DNA was reduced by increasing the gelatin-DNA mixing ratio to attain a saturated value of about 150 nm. The condensed gelatin-DNA complexes showed the zeta potential of 10-15 mV. The cells incubated with the complex exhibited significantly stronger luciferase activities than free plasmid DNA, and the activity was further enhanced by US irradiation. The enhancement was significant for the Sm derivative compared with the corresponding Ed and Sd derivatives. The amount of plasmid DNA internalized into the cells was significantly increased by the complexation with every gelatin derivative, whereas US irradiation did not significantly increase the DNA internalization. US irradiation had no effect on the viability of cells incubated with every gelatin derivative-plasmid DNA complex, although the viability was decreased by the complex incubation. The buffering capacity of Sm derivative was higher than that of Ed and Sd derivatives and comparable with that of polyethylene amine.
Among amine derivatives of gelatin, the Sm derivative enabled the plasmid DNA to induce the US-enhanced gene expression of cells in vitro most effectively because of the superior buffering effect.
本研究旨在检测质粒DNA与胺化明胶衍生物复合物的超声（US）增强基因表达。
用可溶性碳二亚胺制备乙二胺（Ed）、亚精胺（Sd）和精胺（Sm）引入程度不同的明胶衍生物。检测明胶衍生物与质粒DNA复合前后的分子大小和ζ电位。在有或无US照射的情况下与复合物孵育后，测量大鼠胃黏膜细胞的DNA表达，以评估明胶衍生物类型对其基因表达的影响。检测复合物的细胞摄取、细胞活力以及明胶衍生物的缓冲作用。
随着胺化程度的增加，明胶衍生物的表观分子大小和ζ电位增大，尽管胺化程度较高的Sm明胶衍生物的值比其他相应衍生物更大。无论明胶衍生物的类型如何，通过增加明胶 - DNA混合比例可降低质粒DNA的表观分子大小，达到约150nm的饱和值。凝聚的明胶 - DNA复合物的ζ电位为10 - 15mV。与游离质粒DNA相比，与复合物孵育的细胞表现出明显更强的荧光素酶活性，并且US照射进一步增强了该活性。与相应的Ed和Sd衍生物相比，Sm衍生物的增强作用显著。与每种明胶衍生物复合后，内化到细胞中的质粒DNA量显著增加，而US照射并未显著增加DNA内化。US照射对与每种明胶衍生物 - 质粒DNA复合物孵育的细胞活力没有影响，尽管复合物孵育会降低细胞活力。Sm衍生物的缓冲能力高于Ed和Sd衍生物，与聚乙烯胺相当。
在明胶的胺衍生物中，Sm衍生物由于具有优异的缓冲作用，能够使质粒DNA在体外最有效地诱导细胞的US增强基因表达。
