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

本研究的目的是开发一种最佳的药物传递系统，通过提高抗肿瘤药物的靶向效率来增加抗肿瘤效果。通过使用pH敏感的脂质体开发了用于大分子到核的人工载体系统，其可以根据内体内pH的降低来增强脂质体包封的大分子的胞质逃逸。通过共聚焦激光显微镜证实了大分子的胞质递送。此外，通过向分子中添加核定位信号（NLS）来实现大分子的核靶向。由于FITC标记的白蛋白不能被动转运到细胞核，因此通过加入NLS，可以成功地将其靶向到细胞核，这种细胞内大分子调控系统可以作为调控大分子化合物细胞内转运的基本策略。另一方面，已显示己糖激酶II型在肿瘤细胞中特异性转录，并且这可以是待抑制的靶酶。我们的目的是提供DNA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。我们已经成功地建立了一个健全的检测系统，以测量核内DNA的PCR方法。这种方法为我们提供了一个有趣的靶向核DNA和转录活性之间的关系。利用这种新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，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.