阳离子脂质基因载体Lipid-mu peptide-DNA的研究

发布时间：2018-06-05 21:42

本文选题：脂质基因载体 + PEG修饰　；参考：《上海交通大学》2012年博士论文

【摘要】：在过去的二十多年间，基因治疗已经在很多疾病治疗领域将研究从临床前推向了临床，这其中既有单基因疾病比如囊肿性纤维化和杜氏肌营养不良症的治疗，也包括更复杂的疾病比如癌症和心血管疾病的治疗。迄今为止最成功的案例当属重症联合免疫缺陷的基因治疗，不过在许多其他领域基因治疗的进展也非常引人瞩目。虽然在研究的推进过程中发现有的疾病（比如囊肿性纤维化）的基因治疗比预想要困难、见效要慢，使得工业界和学术界对这些疾病基因治疗的关注热度在过去十多年间有所下降，但是基因治疗对于不宜手术的和对传统疗法缺乏响应的疾病仍具有无可替代的优势，结合在多种疾病的临床治疗中取得的成绩，，仍然表明了基因治疗是未来医学的希望之一。载体是基因治疗的重要组成部分，是影响治疗结果的重要因素。因为基因药物本身不能进入细胞，在体内环境中又极易被降解，因此起保护和携载作用的载体会直接影响到基因治疗的效果。纵观进入临床试验的基因药物多是选择使用病毒类基因载体，这是由于病毒类载体具有较高的基因输送效率。但是许多这类临床实验止步于I期或II期，很大程度上是因为病毒类载体的免疫原性和毒性较高。即使有些临床实验采用了ex vivo的策略，即在体外用病毒载体转染细胞，再将细胞通过手术途径植入病灶部位的方法避开人体免疫系统的攻击，病人仍然需要周期性的动手术取出老细胞植入新细胞，这不但增加了手术中感染的风险，也不利于病人接受。而非病毒类载体具有低毒性，低免疫原性，低致癌性的优点，大大提高了使用安全性。除此之外，非病毒类载体对携载基因的大小没有限制，可以重复给药，质量控制简单，制备可重复性高，这些优势都使得非病毒类载体成为未来最终实现基因治疗的可能方式。并且可用于系统给药的非病毒类载体，由于注射方式简单、便捷、微创，最终将成为治疗趋势发展的方向。非病毒类载体成功实现体内基因治疗的前提包括：首先，载体在循环系统中保持稳定以提高在病灶部位的基因药物浓度；其次，载体在靶细胞内及时高效地将基因药物释放到细胞质中以提高转染的效率。本论文按照这两个前提，首先采用高于传统修饰量的PEG脂质对基因载体Lipid-mu peptide-DNA（LMD）进行表面修饰，提高了载体在肿瘤部位的聚集程度；其次使用质子泵抑制剂类药物奥美拉唑抑制细胞中内涵体或溶酶体的酸化过程，提高了载体的体外和体内基因转染效率。具体内容和主要结果总结如下。和普通脂质DNA复合物的制备过程不同，LMD的制备是先将带负电的基因快速加入到带正电的多肽mu中，通过正负电荷相互作用形成带有负电的mu-DNA（MD）粒子，再将MD粒子加入到含有阳离子脂质的脂质体中，再次通过正负电荷相互作用形成净电荷为正电的LMD粒子。整个制备过程可以描述为mu，DNA和脂质体的自发组装，制备耗时短，利于重复。通过对粒径等物理化学性质和体外转染实验的优化，我们确定了LMD三种组分间的最佳比例DNA:mu:cationic lipid是1mg:0.6mg:12μmol。在这个比例下得到MD粒径为102±12nm，zeta电位为-33±1mV；LMD粒径为140±15nm，zeta电位为30±0.3mV。之后我们使用原子力显微镜观察到LMD的形貌是球形或接近球形的粒子，大小比较均一。结合Miller教授实验室的冰冻蚀刻电镜图像，可知LMD具有MD粒子在核心，外面包裹脂质双层的结构。和传统脂质复合物相比，这种结构更加稳定，制备可重复性更高，并且可以在其表面修饰高于传统修饰量（10mol%）的PEG脂质。由于PEG脂质头部的多羟基结构具有良好的亲水性，PEG聚合物长链又可以起到空间位阻的作用，故可以降低被修饰粒子和血液循环中蛋白间相互作用引起的调理作用，减少粒子和带负电蛋白之间聚集而形成大颗粒的程度，增加载体粒子在体内稳定性，延长循环时间，因此PEG修饰的载体也称为长循环载体。我们通过后期孵育法制备了可用于系统给药的长循环PEG-LMD。经过高于传统PEG修饰量（15-25mol%）修饰的LMD的zeta电位由30mV变成接近0，粒径则比修饰前略有增大，为170±30nm。由于实体瘤的高通透性和滞留效应（Enhanced Permeability andRetention，EPR效应）长循环载体可以被动靶向到皮下移植瘤裸鼠的肿瘤部位。较高PEG表面修饰的LMD粒子可通过EPR效应更多地聚集在肿瘤部位，我们通过小动物活体成像、冰冻切片、免疫组化和实时PCR实验分别证实了此结论。使用近红外荧光染料Cy5.5标记LMD的脂质成分，我们使用小动物成像仪对一系列不同PEG表面密度的LMD粒子（0，10，15，23，25，30mol%）在不同时间点于肿瘤处的分布进行了直观的观测：尾静脉注射后，荧光信号很快聚集在肿瘤部位，在较高PEG修饰密度下，比如15%-25%范围内，Cy5.5-PEG-LMD在肿瘤中有较强的荧光信号，在注射5天后，依然可以在肿瘤部位观察到明显的荧光信号，并且肿瘤部位的荧光强度持续高于其他组织。我们进一步从荧光图片上提取出相关组织和肿瘤的荧光强度，用组织面积和全身荧光总强度进行归一化处理，使用非房室模型对数据进行分析，得到了一致的结论。我们通过切片染色的方法用激光共聚焦显微镜观察分别标记了基因物质和脂质成分的LMD在肿瘤的分布，同样观察到含有23mol%PEG的LMD比只有2mol%或10mol%PEG的LMD能够更多地分布在肿瘤部位。并且23%PEG-LMD在不同制剂批次间和不同小鼠体内都表现出很强的瘤内荧光信号。最后我们通过实时PCR对不同PEG修饰密度的LMD在肿瘤部位的分布进行了定量分析，得到相同结论：不同PEG修饰密度的LMD肿瘤含量为25%23%15%30%10%。在优化PEG-LMD体内肿瘤分布后，我们接着考察了载体的基因转染能力。我们使用Luciferase质粒DNA作为报告基因，多轮体外和体内基因转染实验结果显示经过PEG修饰的LMD粒子无法进行有效转染。我们推测造成PEG-LMD转染效率低下的原因可能是PEG脂质阻碍了载体从内涵体中逃逸，随着内涵体pH值降低以及和溶酶体融合，载体粒子中的基因物质最终被酶解而丧失转染能力。为了增加载体从内涵体中逃逸的程度，我们首次将质子泵抑制剂类药物奥美拉唑和基因输送联合应用。基于质子泵抑制剂可以阻碍内涵体和溶酶体中pH值降低，我们假设这一特性可以增加载体粒子从内涵体逃逸的程度。实验证明有质子泵抑制剂存在下，移去荧光标记的23%PEG-LMD6天后依然可以观察到细胞内的荧光，而没有质子泵抑制剂存在下的细胞观察不到明显荧光信号，并且荧光强度和质子泵抑制剂的浓度成正相关关系。体外转染实验表明0.1mg/ml奥美拉唑可将23%PEG-LMD的H1299细胞转染结果提高9倍。当使用碳链较短的PEG脂质DPPE-PEG时，0.1mg/ml奥美拉唑可将23%PEG-LMD的H1299细胞转染结果提高34倍。体内转染实验中150mg/kg奥美拉唑可将23%PEG-LMD对H1299皮下移植瘤的转染效率提高3倍多。实验结果显示奥美拉唑可以通过抑制内涵体和溶酶体pH值降低，增加PEG化脂质载体LMD的体外和体内基因转染效率。
[Abstract]:In the past more than 20 years, gene therapy has been pushed into clinical trials in many areas of disease treatment, including the treatment of single gene diseases such as cystic fibrosis and dystrophy, including more complex diseases such as cancer and heart disease. The most successful cases to date. The genetic treatment of severe combined immunodeficiency, but the progress of gene therapy in many other fields is also very attractive. Although the progress of research has found that some diseases, such as cystic fibrosis, are more difficult than expected and are slower to enable industry and academia to treat these diseases by gene therapy. The heat of concern has declined over the past more than 10 years, but gene therapy still has an irreplaceable advantage for Unoperable and unresponsive diseases of traditional therapies. The achievements in the clinical treatment of various diseases still indicate that gene therapy is one of the hopes of future medicine.
The carrier is an important component of gene therapy, which is an important factor affecting the outcome of the treatment. Because the gene drug itself cannot enter the cell and is easily degraded in the body environment, the carrier of protective and carrying action will directly affect the effect of gene therapy. This is due to the high gene delivery efficiency of viral vectors. However, many of these clinical trials stop at stage I or II, largely due to the high immunogenicity and toxicity of viral vectors. Even some clinical trials have adopted the strategy of ex vivo, that is, transfection of cells with viral vectors in vitro, and then To avoid the attack of the body's immune system by surgical approaches, the patients still need periodic operations to remove the old cells into the new cells, which not only increases the risk of infection in the operation, but also is not conducive to the patient's acceptance, but the non viral vector has the advantages of low toxicity, low immunogenicity and low carcinogenicity. In addition, the non viral vectors have no limitation on the size of the carrying genes, can be repeated to the drug, the quality control is simple, and the preparation is high repeatability. These advantages make the non viral vector a possible way to eventually realize the gene therapy in the future. And it can be used for the non viral vector of the system for drug delivery. Because the injection method is simple, convenient and minimally invasive, it will eventually become the trend of treatment trend.
The precondition of the successful implementation of gene therapy in the body of non viral vectors includes: first, the carrier is stable in the circulatory system to improve the gene drug concentration at the site of the focus. Secondly, the carrier is released into the cytoplasm in time and efficiently in the target cells to improve the efficiency of transfection. First of all, this paper is based on these two prerequisites. The surface modification of the gene carrier Lipid-mu peptide-DNA (LMD) was carried out with the PEG lipid higher than the traditional modifier to improve the concentration of the carrier in the tumor site. Secondly, the acidification process of the endosome or lysosome in the cells was inhibited by omeprazole, the proton pump inhibitor drug, to improve the gene transfection efficiency in vitro and in vivo. The specific content and main results are summarized as follows.
The preparation process of the DNA complex is different from that of the common lipid complex. The preparation of LMD is to quickly add negative electricity to the positive peptide mu, to form a negative mu-DNA (MD) particle by the positive and negative charge interaction, and then add MD particles to the liposomes containing the cationic lipid, and then form a positive and negative charge interaction. The entire preparation process can be described as the spontaneous assembly of mu, DNA and liposomes, and the preparation of the spontaneous assembly of DNA and liposomes is short and beneficial to repetition. By optimizing the physical and chemical properties of the particle size and in vitro transfection experiments, we have determined that the optimum proportion of DNA: mu:cationic lipid between the three components of the LMD is 1mg:0.6mg:12 u mol. in this proportion. When the particle size of MD is 102 + 12NM, zeta potential is -33 + 1mV, LMD particle diameter is 140 + 15nm, and zeta potential is 30 + 0.3mV., we use atomic force microscope to observe that the morphology of LMD is spherical or close to spherical particles, and the size is uniform. The structure is coated with lipid bilayer. Compared with the traditional lipid complex, this structure is more stable, more reproducible, and can modify the PEG lipid on its surface, which is higher than the traditional modification (10mol%).
Because the polyhydroxy structure of the PEG lipid head has a good hydrophilic property, the long chain of PEG can also play the role of space hindrance, so it can reduce the conditioning effect caused by the interprotein interaction between the modified particles and the blood circulation, and reduce the degree of particle and the accumulation of negative proteins to form large particles and increase the carrier particle. The PEG modified carrier is also known as a long cycle carrier in the body, so we have prepared a long cycle PEG-LMD. that can be used for system administration by the later incubation method. The zeta potential of LMD, which is higher than the traditional PEG modification (15-25mol%), is changed from 30mV to nearly 0, and the particle size is slightly larger than that before the modification, which is 170 + 30nm..
Due to the high permeability and retention effect of solid tumor (Enhanced Permeability andRetention, EPR effect) long circulation vector can be passively targeted to the tumor site of nude mice of subcutaneous transplanted tumor. Higher PEG surface modified LMD particles can accumulate more at the tumor site through the EPR effect. We use small animals in vivo imaging, frozen section, immunization This conclusion was confirmed by the histochemical and real-time PCR experiments. Using the near infrared fluorescent dye Cy5.5 labeled LMD lipid components, we used the small animal imager to observe the distribution of a series of LMD particles (0,10,15,23,25,30mol%) with different PEG surface density (0,10,15,23,25,30mol%) at different time points at the tumor. After the injection of the tail vein, the fluorescent letter was used. The number is quickly gathered at the tumor site. At the high PEG modification density, such as the 15%-25% range, Cy5.5-PEG-LMD has strong fluorescence signal in the tumor. After 5 days of the injection, the obvious fluorescence signal can still be observed at the tumor site, and the fluorescence intensity of the tumor site is higher than that of the other tissues. The fluorescence intensity of the related tissues and tumors was extracted, the tissue area and total body fluorescence intensity were normalized, and the data were analyzed with the non atrioventricular model, and a consistent conclusion was obtained. We used laser confocal microscopy to observe the LMD in the tumor by laser confocal microscopy. The distribution of 23mol%PEG was also observed to be more distributed at the tumor site than that of LMD with only 2mol% or 10mol%PEG. And 23%PEG-LMD showed strong intratumoral fluorescence signals between different batches of different preparations and in different mice. Finally, the distribution of LMD at the tumor site by real time PCR on different PEG modification densities was distributed. Quantitative analysis showed that the tumor size of LMD with different PEG density was 25%23%15%30%10%.
After optimizing the tumor distribution in PEG-LMD, we then examined the gene transfection capacity of the carrier. We used the Luciferase plasmid DNA as the reporter gene, and the results of gene transfection experiments in vitro and in vivo showed that the PEG modified LMD particles could not be transfected effectively. The reasons for the low efficiency of PEG-LMD transfection were possible. It is the PEG lipid that prevents the carrier from escaping from the endosome, and with the decrease of the endosome pH and the fusion of the lysosomes, the gene material in the carrier particles eventually loses the transfection capacity. In order to increase the extent of the escape of the carrier from the endosome, the proton pump inhibitor drugs omeprazole and gene delivery are combined for the first time. Proton pump inhibitors can inhibit the reduction of pH values in the endosomes and lysosomes. We assume that this property can increase the degree of escape of the carrier particles from the endosome. Experiments show that the proton pump inhibitors exist in the presence of 23%PEG-LMD6 days after removing the fluorescent labeling, and there are no proton pump inhibitors. No obvious fluorescent signal was observed in the cells below, and the fluorescence intensity was positively correlated with the concentration of proton pump inhibitors. In vitro transfection experiments showed that 0.1mg/ml omeprazole could increase the transfection result of 23%PEG-LMD H1299 cells by 9 times. When the PEG lipid DPPE-PEG of a shorter carbon chain was used, the 0.1mg/ml omeprazole could be the H1299 of 23%PEG-LMD. The results of cell transfection were increased by 34 times. 150mg/kg omeprazole could increase the transfection efficiency of 23%PEG-LMD to H1299 subcutaneous tumor by 3 times. The results showed that omeprazole could decrease the pH value of endosome and lysosome, and increase the gene transfection efficiency of PEG liposome LMD in vivo and in vivo.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R3416

【相似文献】