Department of Drug Delivery Research, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan.
Biomaterials. 2011 Jul;32(20):4659-69. doi: 10.1016/j.biomaterials.2011.03.013. Epub 2011 Apr 9.
The development of gene transfection methods enhancing the level of gene expression under simple and low-toxic condition is required for gene therapy in clinical. Our group has developed the ultrasound (US)-mediated gene transfection method using Man-PEG(2000) bubble lipoplexes, which are US-responsive and mannose-modified gene carriers, and succeeded in obtaining the enhanced gene expression in mannose receptor-expressing cells selectively by the gene transfer using Man-PEG(2000) bubble lipoplexes with US exposure in vitro and in vivo. Here, we investigated pDNA transferring mechanism followed by US exposure to unmodified and Man-PEG(2000) bubble lipoplexes, in particular, focused on US exposure timing. Following investigation of intracellular transferring characteristics, a large amount of pDNA was transferred into the cytoplasm followed by US-mediated destruction of bubble lipoplexes in the gene transfer using both bubble lipoplexes with US exposure. Moreover, the effective gene expression was obtained without TNF-α production when US was exposed until 5 min after the addition of bubble lipoplexes. These findings suggest that the gene transfer using unmodified and Man-PEG(2000) bubble lipoplexes with US exposure enables to transfer pDNA into the cytoplasm, and optimized US exposure timing is important to achieve the high level of gene expression and the low level of pro-inflammatory cytokine production.
需要开发能够在简单、低毒条件下提高基因表达水平的基因转染方法，以实现临床基因治疗。本课题组开发了一种使用 Man-PEG（2000）泡囊脂质体的超声（US）介导的基因转染方法，该方法是一种对 US 有反应且经甘露糖修饰的基因载体，通过使用 Man-PEG（2000）泡囊脂质体与 US 暴露进行基因转移，在体外和体内成功地获得了在甘露糖受体表达细胞中选择性增强的基因表达。在此，我们研究了 US 暴露后未经修饰和 Man-PEG（2000）泡囊脂质体的 pDNA 转移机制，特别关注 US 暴露时机。在研究了细胞内转移特性后，大量的 pDNA 被转移到细胞质中，随后在 US 暴露的两种泡囊脂质体的基因转移中，泡囊脂质体被 US 介导破坏。此外，当 US 暴露时间延长至添加泡囊脂质体 5 分钟后，有效表达基因而不产生 TNF-α。这些发现表明，使用未经修饰和 Man-PEG（2000）泡囊脂质体与 US 暴露的基因转移可以将 pDNA 转移到细胞质中，并且优化 US 暴露时机对于实现高水平的基因表达和低水平的促炎细胞因子产生非常重要。
