肿瘤微酸环境敏感的基因递送系统的构建和初步评价

发布时间：2018-04-11 08:45

  本文选题：pH敏感脂质体 + siRNA　； 参考：《河北医科大学》2014年硕士论文

【摘要】：国际抗癌联盟发布的数据表明，全球癌症发病形式严峻，发病率与死亡率呈持续上升趋势，中国的癌症发病率几乎占了全球的一半，目前我国癌症发病率为285/10万，平均每天每分钟有6人被诊断为恶性肿瘤。在我国，乳腺癌已成为女性最常见的恶性肿瘤之一，目前已步入每年2％～3％的快速增长期。目前的抗肿瘤药物靶向性差、毒副作用大、治疗效果不佳等缺点。 脂质体是一种类似于生物膜结构的双分子层微囊，由于其具有细胞类似结构，进入人体后主要被网状内皮系统吞噬，使药物靶向聚集在肝、脾、肺等组织中。但脂质体作为药物载体仍存在对疾病的靶向性差，稳定性欠佳等缺点。pH敏感脂质体在低pH值时脂质体由有稳定结构变为不稳定结构。使得pH敏感脂质体内吞进细胞后质膜破裂或与其他质膜的融合，这使药物在未达到溶酶体前释放，避免的了溶酶体的降解，更有效的将药物传递到细胞中。由于肿瘤间质液的pH值显著低于周围正常组织，所以研究pH敏感脂质体对肿瘤的治疗作用有很大临床价值。 目的： 由于肿瘤细胞外呈微酸环境，设计了在低pH内可插入细胞膜的多肽pHLIP，通过合成DSPE-PEG-pHLIP。实现对入微酸环境下的肿瘤细胞膜的靶向定位。组氨酸八聚体带正电，可协助小分子进入细胞，本研究中，将硬脂酸和组氨酸八聚体偶合，形成带阳离子的聚合物，由静电吸附作用与siRNA形成SA-H8/siRNA复合物，提高siRNA的载药效率。 方法： 在文献和预实验的基础上，初步确定了pH敏感型聚合物SA-H8的合成方法,在室温条件下，充氮气避光搅拌32h，通过MS对其进行表征。 确定合成导向性脂材（DSPE-PEG-pHLIP）的合成方法，在室温条件下，充氮气避光搅拌48h，通过测定多肽中剩余巯基的含量确定反应终点，并对其进行MS表征。 在文献基础上，采用薄膜分散法制备脂质体，分别以香豆素和siRNA为模型药物，以粒径和包封率为考察指标，确定最佳处方工艺，以制剂的外观、粒径电位和形态作为指标，考察制得脂质体的稳定性。 以人乳腺癌细胞（MCF-7）为细胞模型，分别在pH6.5和pH7.4培养基的环境中，给予压缩载药脂质体和普通载药脂质体，孵育后，将细胞用PBS处理后，用流式细胞仪对载体体外细胞摄取性质进行分析。 结果： 经MS分析，合成得到SA-H8和DSPE-PEG-pHLIP，冷冻干燥后成白色絮状固体,产率分别为48.9%和52.2%；制备香豆素脂质体，所得脂质体粒径较均一，得到微黄色发有乳光的均匀混悬液，并且较稳定，测得该脂质体的包封率为91.0%；制备siRNA的脂质体制剂在透射电子显微镜下均匀分布，粒子中间呈亮光状。 流式细胞分析结果显示，相比普通脂质体，pHLIP修饰载体的细胞摄取具备显著的pH响应特性，，pH6.8条件下摄取明显高于pH7.4环境。 结论： 以香豆素和siRNA为模型药物，以SA-H8阳离子聚合物为siRNA载体，用DSPE-PEG-pHLIP修饰脂质体表面，采用薄膜分散法制备脂质体，稳定性良好。以人乳腺癌MCF-7为细胞模型，pHLIP多肽作为导向性膜材修饰脂质体，在低pH值时，人乳腺癌细胞的摄取率明显高于未经修饰的脂质体，DSPE-PEG-pHLIP靶头对siRNA细胞摄取有促进作用。
[Abstract]:UICC released data show that the serious global cancer incidence rate has continued to rise, and the incidence rate of cancer mortality, Chinese accounted for almost half of the world, China's current cancer incidence rate of 285/10 million, an average of every minute of every day 6 people are diagnosed with malignant tumor in our country, breast cancer it has become one of the most common malignant tumors of women, has entered the year 2% ~ 3% period of rapid growth. The antitumor drug targeting, toxicity, poor treatment and other shortcomings.
Liposome is a kind of similar to the biofilm structure of bilayer microcapsules, because it has the similar structure of the cell, enters the human body mainly by the reticuloendothelial system phagocytic, drug targeting aggregation in the liver, spleen, lung and other tissues. But liposomes as drug carriers still exist for diseases of poor targeting stability the disadvantages of poor.PH sensitive liposome lipid at low pH value by stable structure becomes unstable. The structure of pH sensitive liposome fusion plasma membrane into the cell after swallowing or with other membrane, the drug release has not reached the lysosomes before to avoid lysosomal degradation, will be more effective drug delivery into cells. The tumor interstitial fluid pH was significantly lower than that of the surrounding normal tissue, so the study of therapeutic effect of pH sensitive liposomes on tumor has great clinical value.
Objective:
Because the tumor cells was slightly acidic environment, the design of peptide pHLIP in low pH can be inserted in the cell membrane, through the synthesis of DSPE-PEG-pHLIP. of tumor cell membrane into the acidic environment of the target location. Histidine eight dimers are positively charged, can help small molecules into cells, in this study, the stearic acid and histidine eight polymer coupling, formed with cationic polymer, by electrostatic adsorption and the formation of siRNA SA-H8/siRNA complex, improve the drug loading efficiency of siRNA.
Method锛

本文编号：1735281