Takae Seiji, Miyata Kanjiro, Oba Makoto, Ishii Takehiko, Nishiyama Nobuhiro, Itaka Keiji, Yamasaki Yuichi, Koyama Hiroyuki, Kataoka Kazunori
Department of Materials Engineering, Graduate School of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
J Am Chem Soc. 2008 May 7;130(18):6001-9. doi: 10.1021/ja800336v. Epub 2008 Apr 9.
PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of approximately 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)] micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective endosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.
基于聚乙二醇(PEG)的多聚体胶束能够响应细胞内还原环境而脱离周围的PEG链,被开发用作非病毒基因载体。一种新型的嵌段阳离子聚合物PEG-SS-P[Asp(DET)]的设计如下:(i)在PEG和聚阳离子片段之间插入可生物裂解的二硫键以触发PEG脱离;(ii)基于聚天冬酰胺的阳离子片段,带有侧翼N-(2-氨基乙基)-2-氨基乙基团,即P[Asp(DET)],其中Asp(DET)单元作为缓冲部分,以最小的细胞毒性诱导内体逃逸。由PEG-SS-P[Asp(DET)]和质粒DNA(pDNA)形成的多聚体胶束在水性介质中稳定分散,由于形成亲水性PEG栅栏,尺寸范围狭窄,约为80nm,而在化学计量电荷比下加入10mM二硫苏糖醇(DTT)时会发生聚集,这表明PEG通过二硫键断裂从胶束上脱离。与没有二硫键的PEG-P[Asp(DET)]胶束相比,PEG-SS-P[Asp(DET)]胶束显示出基因转染效率高1-3个数量级,基因表达起始更快,这是因为基于内体中PEG脱离的内体逃逸更有效。这些发现表明,PEG-SS-P[Asp(DET)]胶束作为一种非病毒基因载体,可能具有在规定时间发挥高转染作用且细胞毒性最小的潜在应用前景。