新型药物递送系统LTV-HR与酸敏感性穿膜肽LH-1、LH-2的设计、合成以及活性测定

发布时间：2018-03-02 05:00

本文关键词： 酸敏感性细胞穿膜肽药物递送系统肿瘤靶向性　出处：《兰州大学》2017年硕士论文　论文类型：学位论文

【摘要】：传统的肿瘤治疗方案中化学治疗具有严重的毒副作用,因为抗肿瘤药物具有渗透性差以及不能特异性生物分布的特点。为了克服抗肿瘤药物的这些缺点,高渗透性、靶向性、安全性的载体的研究发现受到业界的广泛关注。现在细胞穿膜肽被认为是最有潜力的药物载体之一。它具有快速大量进入细胞内以及低毒的特点,但是天然的细胞穿膜肽不能区分正常组织和肿瘤组织。目前提出的靶向修饰策略分为两种:一种是靶向到肿瘤部位微环境,因为肿瘤部位的低pH值、基质金属蛋白酶、乏氧以及脉管系统与正常组织部位的存在差异;一种是受体与配体靶向策略,研究者们发现许多肿瘤细胞表面存在过表达的受体,而正常细胞则不存在或者低表达。本论文研究的第一部分我们设计了一种新的药物递送系统。课题组以前设计出了一条酸敏感性穿膜肽HR-1,它具有在生理条件下穿膜活性低而在酸性条件时却拥有高穿膜活性的特点。但HR-1的靶向为被动靶向,为了增强HR-1携带抗肿瘤药物进入细胞的能力,我们决定将酸敏感性肽HR-1与主动靶向肽LTV通过半胱氨酸连接得到新的药物递送系统LTV-HR,其中LTV能够主动靶向到乳腺癌细胞上过表达受体HER2。LTV-HR在与喜树碱(CPT)通过二硫键共价结合,得到LTV-CPT-HR。在激光共聚焦、细胞凋亡实验和抗肿瘤活性实验中我们发现药物递送系统LTV-HR具有很好的酸敏感性,它在低酸条件pH6.0时携带了更多的喜树碱进入癌细胞中,但是在拮抗实验中我们发现LTV-HR中的靶向肽LTV并没有显示出很明显的主动靶向作用。在体外实验中,我们发现不管是与细胞长时间共孵育或在不同pH条件下的共孵育以及乳酸脱氢酶释放实验中LTV-HR皆展示了安全低毒的特点。但在溶血活性实验中,在高浓度200μmol/L时溶血率达到了13.35%,但是100μmol/L时即为测试最大浓度40μmol/L的2.5倍时溶血率为3.44%,说明在测试浓度范围内LTV-HR是安全的。综上所述,LTV-HR具有很好的酸敏感性,同时具有低毒性的特点,它有望成为肿瘤靶向治疗的载体之一。本论文研究的第二部分我们设计了两种新的酸敏感性穿膜肽。我们对母肽LK(LKKLLKLLKKLLKL-NH2)进行氨基酸替换改造得到了肽LH-1与LH-2。在细胞摄取实验中我们看到LH-2显示出了明显的酸敏感性,它在pH6.0条件下的荧光强度显著高于pH7.4条件,说明在酸性条件下LH-2大量的被细胞摄取。而LH-1在两个pH条件下细胞摄取没有明显区别。在毒性实验中,我们发现LH-1、LH-2与母肽LK相比毒性都显著降低。在不同pH条件肽毒性实验与LDH释放实验中LH-2显示毒性皆很弱但LH-1显示出了差异性,在pH7.4时LH-1的细胞存活率和LDH释放率显著低于pH6.0条件,说明LH-1在pH7.4时正电荷被屏蔽所以毒性作用表现很低,而在pH6.0时LH-1中组氨酸被质子化带上大量正电荷从而表现出和母肽相似的毒性作用。综合毒性实验以及细胞摄取实验的结果,我们选择酸敏感性好且毒性很低的LH-2与喜树碱结合做抗肿瘤活性实验,MTT数据显示LH-2在pH6.0时拥有更强的穿膜效应,携带了更多的喜树碱进入细胞内杀死癌细胞,与母肽LK相比LH-2不仅毒性显著降低同时对于肿瘤部位酸性微环境拥有了靶向作用。
[Abstract]:Chemical treatment has serious side effects of traditional cancer therapy, because of antitumor drugs with poor permeability and specific biological distribution characteristics. In order to overcome these shortcomings of anti-tumor drugs, high permeability, targeted research, the safety of carrier that has received widespread attention in the industry. In the cell peptide is now considered one of the most promising drug carrier. It has rapidly entered the cells and low toxicity characteristics, but natural CPPs can not distinguish between normal and tumor tissue. The targeted modification strategy is divided into two types: one is targeted to the tumor microenvironment because parts of the tumor, low pH, matrix metalloproteinase, differences in hypoxia and vascular system and normal tissue; a receptor and ligand targeting strategies, the researchers discovered that many tumor cells There is overexpression of the receptor, while normal cells do not exist or low expression. In the first part of this thesis we design a new drug delivery system. The research group had previously designed an acid sensitive membrane penetrating peptide HR-1, it has the characteristics of penetrating activity under physiological conditions and low in acidic conditions it has high penetrating activity. But the target for passive targeting HR-1, in order to enhance the HR-1 carrying anticancer drugs into cells, we decided to put the acid sensitive peptide HR-1 and active targeting peptide LTV by cysteine connection get a new drug delivery system LTV-HR, which can active targeting to LTV expression a receptor HER2.LTV-HR in breast cancer cells with camptothecin (CPT) by combining the two disulfide covalent LTV-CPT-HR. in laser confocal microscopy, cell apoptosis assay and anti tumor activity of the experiment we found that the drug delivery system LTV-HR Has good acid sensitivity, it is in low acid conditions pH6.0 carrying more camptothecin into cancer cells, but in the antagonistic experiment we found LTV-HR targeting peptide LTV showed no obvious effect to the active target. In vitro experiment, we found that regardless of cell with long time co cultured or co incubated in different pH conditions and activities of lactate dehydrogenase release experiments of LTV-HR are demonstrated the characteristics of safety and low toxicity. But in hemolytic experiment, in high concentration of 200 mol/L when the hemolysis rate reached 13.35%, but 100 mol/L which is 2.5 times the concentration of 40 mol/L test the hemolysis rate was 3.44%, indicated in the test concentration range of LTV-HR is safe. In conclusion, LTV-HR has good acid sensitivity, but also has the characteristics of low toxicity, it is expected to become the carrier of targeted tumor therapy. This paper studies second We designed two kinds of acid sensitivity of new membrane penetrating peptide. We of the parent peptide LK (LKKLLKLLKKLLKL-NH2) of amino acid substitution transformation to get the peptide LH-1 and LH-2. in cellular uptake experiments we see LH-2 showed obvious acid sensitivity, its fluorescence under pH6.0 intensity was significantly higher than that of pH7.4, indicating uptake LH-2 under acidic conditions is a large amount of cells. LH-1 in the two pH under the condition of cell uptake. No obvious difference in the toxicity experiment, we found that LH-1 and LH-2 toxicity were significantly decreased compared with the parent peptide. LK in different pH peptide toxicity test and LDH assay in LH-2 showed toxicity are very weak but LH-1 show the difference in pH7.4 LH-1, the cell survival rate and LDH release rate was significantly lower than that of pH6.0, indicated that the LH-1 in pH7.4 when the positive charge is shielded so the toxic effect is very low, and LH-1 in pH6.0 by histidine Take a large number of protonated positive charge can exhibit toxicity and similar results. The parent peptide toxicity test and cell uptake experiments, we choose LH-2 and acid sensitivity of camptothecin and low toxic combination of anti tumor activity, MTT data show that LH-2 has stronger penetrating effect in pH6.0, carrying more camptothecin into cells to kill cancer cells, compared with the parent peptide LK LH-2 not only significantly reduce the toxicity and for tumor acidic microenvironment have targeting effect.

【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R914

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