新型PEI衍生物材料的合成及在肿瘤基因递送中的应用研究

发布时间：2018-05-02 20:41

本文选题：聚乙烯亚胺（Polyethyleneimine + PEI）　；参考：《吉林大学》2014年博士论文

【摘要】：近年来，恶性肿瘤已经成为威胁人类生命健康的主要杀手，我国每年因恶性肿瘤而死亡的人数达到220万，占我国居民死亡原因的第一位。因此，恶性肿瘤的治疗已经成为亟待解决的世界性难题，具有极其重要的战略意义。现如今，临床常用的肿瘤治疗手段包括手术、化疗和放疗等，手术只能使一部分肿瘤患者受益，而化疗和放疗对正常组织器官毒副作用大，严重影响患者的生活质量。此外，长期使用化学药物治疗肿瘤，会产生多药耐药现象（Multidrug resistance，，MDR），降低了化疗药物的治疗效果。而与传统治疗手段相比，基因治疗有望从根本上治疗疾病，已成为目前国际学术界研究的焦点，它被认为是医学和药学领域的一次革命，是当今生物医学发展中最重要的里程碑之一，同时也必将对传统制药业产生深远影响和冲击，有望成为未来在肿瘤临床治疗中的“新宠”。基因治疗主要有三个方面：基因药物、基因诊断和基因传输。随着人类基因组计划（human genome project, HGP）的成功实施，基因诊断和基因药物的制备不再是基因治疗的障碍；但如何安全、有效的将药物基因运载到靶细胞并起到治疗效果，成为获得基因治疗良好效果的瓶颈之一。常见的基因递送方式可以分为物理方法、化学方法和生物方法。化学方法和生物方法都是利用载体承载基因药物，载体的地位极其重要，也成了基因治疗发展中的关键性问题。生物学上根据来源将基因载体分为质粒载体和病毒载体；而化学研究人员习惯将质粒看做DNA的一种，而将载体分为病毒类载体（ViralVector）和非病毒类载体（Non-viral Vector）。目前在临床使用中应用最多的仍然是病毒类载体，部分转染效率可高达90%以上，但病毒类载体存在严重的免疫源性等自身难以克服的缺陷。相对而言，非病毒类载体由于其特有的优势而越来越受到关注。聚乙烯亚胺(Polyethyleneimine, PEI)分子已经成为了非病毒类基因载体领域里发展最快、最具有研究价值和应用潜质的一类基因传输载体。对于转染效率而言，理想的PEI分子量范围约为11.9×103~70.0×103，其中重均分子量为25K的超支化PEI（PEI25K）转染效率最高，因而成为衡量非病毒基因载体各项性能的“黄金标准”。PEI独特的“质子海绵效应”有利于实现细胞内的内涵体逃逸。然而，PEI25K作为非病毒基因载体材料仍然有很多缺陷，如相对较低的基因转染效率、细胞毒性大、聚合物结构的不可降解性等。因此，对其进行结构改造就显得尤为必要。本论文立足于肿瘤基因治疗这一当前研究热点，设计了两类新型PEI衍生物材料，我们首先将不同数量的带有疏水基团的N-乙酰-亮氨酸，以EDC/NHS催化，使其与PEI25K的伯氨基进行偶联，来研究N-乙酰-亮氨酸引入的数量对PEI25K作为基因传递材料能力的影响。然后在前期工作基础上，进行了N-异丙基丙烯酰胺改性PEI25K基因载体的制备及性能研究，通过一系列的化学及细胞实验对衍生物的材料结构、细胞毒性、核酸结合能力、基因转染效率进行研究，并采用改性后的PEI衍生物（PEN）将外源的p53基因递送到p53基因缺失的前列腺癌PC-3细胞系与p53野生型细胞系Hela中进行对比研究。第一部分：N-乙酰-亮氨酸(N-Ac-Leu)改性PEI25k基因载体的构建与评价 1.本章采用EDC/NHS催化反应合成了三种N-Ac-Leu修饰的PEI衍生物，分别为N-Ac-Leu-PEI (60:1)、 N-Ac-Leu-PEI (80:1)、N-Ac-Leu-PEI (100:1)并利用核磁共振氢谱（1H NMR）对聚合物的结构进行表征。 2.通过凝胶电泳可以表明，三种N-Ac-Leu-PEI聚合物均能很好的与pEGFP质粒结合，但结合能力低于未修饰的PEI25K。通过表面电位测定可以得知三种N-Ac-Leu-PEI的表面电位在+30mV，恰好可以说明N-Ac-Leu-PEI与质粒结合能力的降低。粒径测定结果表明N-Ac-Leu-PEI聚合物的粒径在180~220nm范围内。 3. N-Ac-Leu-PEI基因载体材料的溶血实验证明载体材料溶血效应相对于PEI25K明显降低，具有很好的血液相容性；蛋白吸附实验中，N-Ac-Leu-PEI蛋白吸附值相对于PEI25K明显降低，且随时间的增加蛋白吸附值缓慢降低。由此证明N-Ac-Leu基团的引入可改善PEI的生物相容性，可望用于活体实验中。 4. MTT实验结果表明，N-Ac-Leu-PEI在HeLa细胞中的细胞毒性较PEI25K有明显降低。 5.三种N-Ac-Leu-PEI聚合物均能有效的介导基因转染，且转染能力高于PEI25K。N-Ac-Leu-PEI (100:1)在质量比为3.0的条件下转染效率最高。第二部分：PEI衍生物基因载体的设计制备以及性能研究 1.首先在PEI骨架上偶联上N-异丙基丙烯酰胺，然后通过细化投料比，得到五种PEI衍生物（PEN）。使用凝胶阻滞实验和细胞毒性试验初步筛选出一种载体。之后通过核磁共振氢谱对所选材料的结构进行了表征。 2.使用纳米粒度电位仪检测了载体、质粒以及载体质粒复合物的粒径大小以及表面电位。 3.通过MTT实验检测了载体的细胞毒性，结果显示，修饰后的载体毒性低于PEI。 4. DNase I酶降解实验发现，在载体质粒质量比不小于0.8时，载体就能够实现对DNA的完全保护。相比较而言， PEN对DNA的保护能力较PEI略有下降。 5.通过绿色荧光蛋白质粒转染实验和PGL-3质粒转染实验，分别定性定量考察了材料作为基因载体的传递性能。实验数据表明，在PEI上引入N-异丙基丙烯酰胺，降低了聚合物的表面电荷，并引入了一定量的疏水基团。但是疏水基团的过多引入反而会降低载体的基因转染效率。在定性转染试验中，改变载体的加入量发现，在载体质粒质量比为2时，转染效果最好。在定量转染实验中，PEI展现了不错的转染效率，PEN虽然不及PEI，但和商业转染试剂相比，转染效率相差无几。 6.在前列腺癌细胞系PC-3中，通过RT-PCR和Western blot分别从mRNA水平和蛋白水平证明p53基因的表达。之后通过检测相关蛋白表达的变化证明表达出的p53蛋白的确具有正常的功能。之后通过检测caspase家族蛋白活性和线粒体膜电位变化辅助说明上述问题。 7.使用流式细胞仪检测周期阻滞和细胞凋亡情况，在检测周期阻滞时发现Hela细胞系不存在周期阻滞现象，通过DAPI染色，证明细胞存在凋亡现象。 8.综合各方面的实验结果，证明经过修饰，虽然在一定程度上降低了PEI的基因转染效率，但所得到的衍生物的毒性低，而且其基因转染效率可以和商业转染试剂相媲美。而且在递送p53基因致使癌症细胞凋亡的试验中，展现了较好的转染性能，后续的凋亡相关实验也证明外源的p53基因的确可以代替或者补偿内源性缺失或者无效的p53基因。
[Abstract]:In recent years, malignant tumors have become the main killer of human life and health. The number of deaths from malignant tumors in China is 2 million 200 thousand, which accounts for the first cause of death in our country. Therefore, the treatment of malignant tumors has become a worldwide problem to be solved urgently. It is of great strategic significance. Now, it is commonly used in clinical practice. The treatment of cancer includes surgery, chemotherapy and radiotherapy, and the operation can only benefit a part of the tumor patients. Chemotherapy and radiotherapy have large side effects on normal tissues and organs, which seriously affect the quality of life of the patients. In addition, the long-term use of chemical drugs in the treatment of tumors can produce multidrug resistance (Multidrug resistance, MDR) and reduce the chemotherapy. Compared with the traditional treatment, gene therapy is expected to cure disease fundamentally. It has become the focus of international academic research. It is considered as a revolution in the field of medicine and pharmacy. It is one of the most important milestones in the development of biomedicine, and it will also have a far-reaching effect on the traditional pharmaceutical industry. Impact and impact is expected to become the "new favorite" in clinical treatment of cancer in the future.
Gene therapy has three main aspects: gene medicine, gene diagnosis and gene transmission. With the successful implementation of the human genome project (human genome project, HGP), gene diagnosis and the preparation of gene drugs are no longer a barrier to gene therapy; however, how to safely, effectively carry the drug genes to the target cells and play a therapeutic effect, One of the bottlenecks in the good effect of gene therapy is that the common way of gene delivery can be divided into physical, chemical and biological methods. Both chemical and biological methods use carriers to carry genetic drugs. The status of carriers is extremely important, and it is also a key problem in the development of gene therapy. Biological sources will be based on the source. Gene vectors are divided into plasmid vectors and viral vectors, and chemical researchers are used to treat plasmids as one of DNA and divide the vectors into viral vectors (ViralVector) and non viral vectors (Non-viral Vector). The most used in clinical use are viral vectors, and the partial transfection efficiency can be as high as 90%. Polyethyleneimine (PEI) has become more and more popular in the field of non viral gene carriers, which have become the fastest, most valuable and applied potential in the field of non viral gene carriers. Gene transmission carrier.
For the transfection efficiency, the ideal PEI molecular weight range is about 11.9 x 103~70.0 x 103, and the hyperbranched PEI (PEI25K) transfection efficiency is the highest with the weight average molecular weight 25K. Thus, the "gold standard".PEI unique "proton sponge effect" is beneficial to the realization of intracellular endosomal escape. However, as a non viral gene carrier, PEI25K still has many defects, such as relatively low gene transfection efficiency, large cytotoxicity, and non degradable polymer structure. Therefore, it is particularly necessary to reconstruct its structure.
This paper, based on the current research hotspot of tumor gene therapy, designs two new types of PEI derivatives. First, we catalyze N- acetyl leucine with different numbers of hydrophobic groups, catalyze it with EDC/NHS, and study the number of N- acetylleucine introduced to PEI25K as a gene transfer. On the basis of previous work, the preparation and performance of N- isopropylacrylamide modified PEI25K gene carrier was studied on the basis of previous work. Through a series of chemical and cell experiments, the material structure, cytotoxicity, nucleic acid binding capacity and gene transfection efficiency of the derivatives were studied, and the modified PEI derivatives (P) were used. EN) the exogenous p53 gene was delivered to the p53 gene deficient prostate cancer PC-3 cell line and p53 wild-type cell line Hela.
Part one: Construction and evaluation of N- acetylleucine (N-Ac-Leu) modified PEI25k gene vector.
1. in this chapter, three kinds of N-Ac-Leu modified PEI derivatives were synthesized by EDC/NHS catalytic reaction, which were N-Ac-Leu-PEI (60:1), N-Ac-Leu-PEI (80:1), N-Ac-Leu-PEI (100:1) and the structure of the polymer was characterized by NMR (1H NMR).
2. the gel electrophoresis shows that the three N-Ac-Leu-PEI polymers can be well combined with pEGFP plasmids, but the binding ability is lower than the unmodified PEI25K. by the surface potential measurement. The surface potential of the three N-Ac-Leu-PEI can be found in +30mV, which shows the decrease of the binding capacity of N-Ac-Leu-PEI and plasmids. The results of particle size determination show that The particle size of N-Ac-Leu-PEI polymer is in the range of 180~220nm.
The hemolysis test of the 3. N-Ac-Leu-PEI gene carrier material showed that the hemolysis effect of the carrier material was significantly lower than that of the PEI25K and had a good blood compatibility. In the protein adsorption experiment, the adsorption value of N-Ac-Leu-PEI protein was significantly lower than that of the PEI25K, and the adsorption value of the protein decreased slowly with the increase of time. Thus, the N-Ac-Leu group was proved to be introduced. It can improve the biocompatibility of PEI and is expected to be used in vivo experiments.
4. MTT test results showed that the cytotoxicity of N-Ac-Leu-PEI in HeLa cells was significantly lower than that in PEI25K.
5. the three N-Ac-Leu-PEI polymers all can effectively mediate gene transfection, and the transfection efficiency is higher than that of PEI25K.N-Ac-Leu-PEI (100:1) with the highest transfection efficiency under the condition of mass ratio of 3.
The second part: design, preparation and properties of PEI derivative gene vector.
1. first, N- isopropyl acrylamide was coupled on the PEI skeleton, and then five PEI derivatives (PEN) were obtained by refining the feeding ratio. A carrier was screened by gel block experiment and cytotoxicity test. The structure of the selected materials was characterized by NMR.
2. the particle size and surface potential of vectors, plasmids and plasmid plasmids were detected by nanoparticle potentiometer.
3. the cytotoxicity of the carrier was detected by MTT test. The results showed that the toxicity of the modified vector was lower than PEI..
The 4. DNase I enzyme degradation experiment showed that the carrier could fully protect the DNA when the plasmid mass was less than 0.8. Compared with the PEI, the protection ability of DNA was slightly lower than that of PEI.
5. through green fluorescent protein plasmid transfection and PGL-3 plasmid transfection experiments, the materials were qualitatively and quantitatively investigated.
As the transmission performance of the gene carrier, the experimental data show that the introduction of N- isopropyl acrylamide on PEI reduces the surface charge of the polymer and introduces a certain amount of hydrophobic groups. But the excessive hydrophobic group will reduce the gene transfection efficiency of the carrier. When the plasmid mass ratio was 2, the transfection efficiency was the best. In the quantitative transfection experiment, PEI showed a good transfection efficiency, while PEN was not as good as PEI, but the transfection efficiency was similar to that of commercial transfection reagents.
6. in the prostate cancer cell line PC-3, the expression of the p53 gene was demonstrated by RT-PCR and Western blot respectively from the mRNA level and protein level. Then the expression of the expressed protein was proved to have normal function by detecting the changes in the expression of the associated protein. Then, the changes in the activity of caspase family protein and the changes of mitochondrial membrane potential were detected. The above question.
7. the flow cytometry was used to detect the cell cycle arrest and cell apoptosis. The Hela cell line was found to have no periodic block during the detection period, and the cell apoptosis was demonstrated by DAPI staining.
8. the experimental results showed that, although the transfection efficiency of PEI was reduced to a certain extent, the toxicity of the derived derivatives was low, and the gene transfection efficiency was comparable to that of commercial transfection reagents. Moreover, the transfection efficiency of the gene transfection was better than that of the commercial transfection reagent. The subsequent apoptosis related experiments also showed that the exogenous p53 gene could replace or compensate for endogenous deletion or ineffective p53 gene.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R318.08;R73-36

【参考文献】