Optimization of drug delivery system for antitumor agents based on the energy metabolism inhibition of tumor cells

本研究的目的是通过提高抗肿瘤药物的靶向效率，开发一种最佳的给药系统来提高抗肿瘤效果。利用pH敏感脂质体构建了大分子到细胞核的人工载体系统，该系统可以根据内体pH值的降低增强脂质体包裹的大分子的胞质逃逸。用激光共聚焦显微镜证实了大分子的胞质传递。此外，通过在分子中加入核定位信号（NLS），实现了大分子的核靶向。fitc标记的白蛋白不能被动传递到细胞核，通过添加NLS成功靶向细胞核。这种细胞内控制大分子的系统可以成为操纵高分子量化合物在细胞内运输的基本策略。另一方面，已经证明ii型己糖激酶在肿瘤细胞中特异性转录，这可能是一个被抑制的靶酶。我们的目标是传递DNA来抑制肿瘤细胞中己糖激酶ii的转录。为了实现这一点，需要定量分析系统来测量细胞内细胞器（如细胞核）中的DNA。我们成功地开发了一套完善的核内DNA pcr检测系统。这种方法为我们提供了目标核DNA和转录活性之间有趣的关系。利用这种新开发的细胞内调控系统和定量分析方法，我们将能够优化合理的药物载体系统来抑制肿瘤细胞的转录。

The objective of this study is to develop an optimum drug delivery system to increase antitumor effect by enhancing targeting efficiency of antitumor agents. An artificial carrier system for macromolecules to the nuclei was developed by using a pH-sensitive liposomes, which can enhance cytosolic escape of liposomally encapsulated macromolecules depending on the intra-endosomal pH decrease. The cytosolic delivery of macromolecules was confirmed by confocal laser microscopy. In addition, nuclear targeting of macromoleules was achieved by adding a nuclear localization signal (NLS) to the molecules. The FITC-labelled albumin, which can not be passively delivered to nucleus, was successfully targeted to nucleus by adding NLS.This system for intracellular control of macromolecules can be a basic strategy to manipulate intracellular trafficking of high molecular weight compounds. On the other hand, it has been shown that hexokinase type-II is specifically transcripted in tumor cells and this can be a target enzyme to be inhibited. We aimed to deliver DNA to inhibit the transcription of hexokinase-II in tumor cells. To achieve this, the quantitative assay system was required to measure DNA in intracellular organella such as nucleus. We have succeeded to develop a sound assay system to measure intra-nuclear DNA by applying PCR-method. This method provided us an interesting relationship between targeted nuclear DNA and transcription activity. By using this newly developed intracellular regulation system and quantitative assay method, we will be able to optimize rational drug carrier system to inhibit transcription in tumor cells.