阳离子修饰的PEG化聚乳酸纳米基因载体的实验研究

发布时间：2018-04-23 14:35

本文选题：聚乙二醇(PEG) + 聚乳酸(PLA)　；参考：《中南大学》2007年硕士论文

【摘要】： 近年来，随着基因转移技术的日趋成熟，基因治疗成为生物科学和临床医学的研究热点之一。基因治疗的载体问题，以及载体相关的免疫反应、细胞毒性与安全性问题是限制基因治疗发展和临床应用的瓶颈。目前基因治疗的研究和临床应用中常用的载体分为病毒载体和非病毒载体，各个载体均有各自的优缺点，病毒载体主要的潜在缺点包括：①致病可能性；②免疫原性；③大规模生产的困难性，而非病毒导入系统正受到更多的关注，它的主要的优点包括安全，容易大规模生产等，主要缺点是不能获得较高水平的基因导入和表达。纳米粒基因转运体是近年发展起来的一种新型的非病毒基因转运载体。它是将DNA、RNA等基因治疗分子包裹在纳米颗粒之中或吸附在其表面，同时也在颗粒表面耦联特异性的靶向分子，如特异性配体、单克隆抗体等，通过靶向分子与细胞表面特异性受体结合，在细胞摄取作用下进入胞内，实现安全有效的靶向性基因治疗。纳米粒材料的选择是成功进行纳米基因转运和治疗的关键。所选择的材料必须是生物可降解型或者易于从体内排泄，而不产生有害的降解产物，且无免疫原性，不引起机体的免疫排斥反应。高分子生物降解材料制备的纳米颗粒具有稳定、无毒、无抗原性、生物相容性好、对所转运基因的表达有缓释作用及对基因有保护作用等优点，是良好的纳米基因转运载体材料。研究证实，纳米粒与DNA的连接多通过静电吸引作用来完成。DNA的磷酸骨架所带的负电荷只能与表面带正电性的载体结合。因此，需要利用生物活性分子对纳米颗粒的表面进行改性，使其表面携带阳离子物质，起到防止颗粒团聚，并有利于结合DNA分子的作用。目的：构建一种理想的纳米基因载体转运系统，使其能有效的保护转运基因不被核酶降解，并具备较高的基因转染效率、有效的输送基因至靶位点以及良好的生物相容性。方法：在本研究中，基于我们的前期工作，首次创新性的选择单甲氧基聚乙二醇-聚乳酸(MePEG-PLA)和壳聚糖(chitosan，CS)通过透析过滤法来制备MePEG-PLA-CS纳米粒作为基因转运载体，并用正交实验设计方法安排实验，优选制备工艺条件。使用AFM(原子力显微镜)和Zetasizer(Marlven，粒径分析仪)来检测MePEG-PLA-CS纳米粒和MePEG-PLA-CS／DNA复合物的Zeta电位以及粒径分布。我们通过静电吸附作用将MePEG-PLA-CS纳米粒和带正电荷的DNA的结合在一起形成MePEG-PLA-CS／DNA复合物，通过分光光度计法来检测MePEG-PLA-CS／DNA复合物中DNA的浓度，从而计算DNA的结合效率。通过凝胶阻滞实验和DNaseI消化实验来检测该基因载体转运系统对DNA的保护能力。采用MTT法研究MePEG-PLA-CS纳米粒对人肝癌细胞(HepG2)及正常肝细胞(L-02)的影响。使用MePEG-PLA-CS携带pEGFP-C1(绿色荧光蛋白质粒)pDNA作为报导基因转染COS7细胞来评价体外转染效率，其中商业化脂质体(Lipofectamine2000，Invitrogen)作为阳性对照而裸DNA作为阴性对照。进一步的，构建BALB／C瘤鼠模型，采用MePEG-PLA-CS纳米粒载报告质粒绿色荧光蛋白(EGFP)研究体内基因转染的能力。结果：通过正交实验，我们得到了制备MePEG-PLA-CS NP的最优条件，AFM和粒径分析仪证明该纳米粒表面平滑完整，分散良好，无粘附团聚现，，其表面电位为正，该纳米基因载体转运系统DNA结合效率(DNA loading efficiency)为91％±4.5％。MePEG-PLA-CS纳米粒由于表面带正电荷而获得结合带负电DNA的能力。同时，实验也证实，MePEG-PLA-CS纳米颗粒／DNA复合物能保护所携带的DNA免受核酸酶的降解。本研究发现，MePEG-PLA-CS纳米颗粒对正常的肝细胞(L-02)在一定的剂量范围内无细胞毒性，只在高浓度下才会表现出一定的细胞毒性作用。在体外基因转运中，该纳米颗粒可有效转运EGFP(绿色荧光蛋白)报道基因表达质粒进入COS7细胞，其转运效率达40％，强于相同条件下脂质体的转染效率。在体内基因转染实验中，由MePEG-PLA-CS纳米粒处理过的裸鼠一组，在肿瘤组织中EGFP基因表达最高，这证实了MePEG-PLA-CS纳米粒借助PEG能够在循环系统中长期滞留而不被网状内皮系统捕获，通过EPR(选择性滞留效应)效应富集到肿瘤组织中，从而具有靶向治疗肿瘤的能力。结论：采用透析过滤法，优化工艺条件，成功制备粒径较小、分布均匀的聚MePEG-PLA-CS纳米颗粒。使该纳米颗粒表面带正电荷从而结合质粒DNA，组装成MePEG-PLA-CS纳米颗粒基因转运体系。通过体内外基因转染及保护DNA等试验，证实该转运体系可保护所携带DNA免受核酸酶的降解，并且是一种低毒高效的纳米基因载体，我们的研究为应用纳米粒来实现肿瘤的基因治疗奠定了良好的科学基础。
[Abstract]:In recent years, with the growing maturity of gene transfer technology, gene therapy has become one of the hotspots in biological science and clinical medicine. The problem of gene therapy carrier, carrier related immune response, cytotoxicity and safety are bottlenecks restricting the development and clinical application of gene therapy. The research and clinical study of gene therapy at present The commonly used carriers are divided into virus carriers and non viral vectors. Each carrier has its own advantages and disadvantages. The main potential shortcomings of the virus carrier include: (1) the possibility of disease; (2) immunogenicity; (3) the difficulty of large-scale production; and the non virus introduction system is being paid more attention, and its main advantages include safety and ease. Large scale production, such as mass production, is the main disadvantage of not obtaining high level of gene introduction and expression. The nanoparticle gene transporter is a new type of non viral gene transport carrier developed in recent years. It is DNA, RNA and other gene therapy molecules wrapped in nanoparticles or adsorbed on its surface, but also on the surface of particles. Targeted molecules, such as specific ligands, monoclonal antibodies, are combined with specific receptors on the cell surface by targeting molecules and enter the cell under cell uptake to achieve a safe and effective targeted gene therapy.
The selection of nanoparticles is the key to the successful transport and treatment of nanoscale genes. The selected materials must be biodegradable or excreted easily from the body without producing harmful degradation products, without immunogenicity, and no immune rejection. The nanoparticles prepared by polymer biodegradable materials are stable. It is a good nanoscale transport carrier material, which has the advantages of non-toxic, antigenicity and biocompatibility. It is a good nanoscale transport carrier material for the expression of the transporter gene and the protective effect on the gene. It is confirmed that the connection of the nanoparticles and DNA is mostly through the electrostatic attraction to complete the negative charge of the phosphoric acid skeleton of.DNA only with the positive surface of the surface. Therefore, it is necessary to use bioactive molecules to modify the surface of the nanoparticles to carry the cationic substance on the surface, which prevents the agglomeration of particles and is beneficial to the interaction of DNA molecules.
Objective: to construct an ideal nanoscale carrier transport system, which can effectively protect the transport genes from ribozyme degradation, and have high gene transfection efficiency, effective gene delivery to target loci and good biocompatibility.
Methods: in this study, based on our previous work, the first innovative selection of mono methoxy poly (MePEG-PLA) and chitosan (chitosan, CS) was used to prepare MePEG-PLA-CS nanoparticles as a gene transport carrier by dialysis filtration method, and the experiment was arranged by orthogonal test design method, and the preparation conditions were optimized. AF M (atomic force microscopy) and Zetasizer (Marlven, particle size analyzer) are used to detect the Zeta potential and particle size distribution of MePEG-PLA-CS nanoparticles and MePEG-PLA-CS / DNA complexes. By electrostatic adsorption, we combine MePEG-PLA-CS nanoparticles with a positive charge DNA together to form a MePEG-PLA-CS / DNA complex, through a spectrophotometer. The method was used to detect the concentration of DNA in the MePEG-PLA-CS / DNA complex and calculate the binding efficiency of DNA. The protective ability of the gene carrier transport system to DNA was detected by gel block experiment and DNaseI digestion experiment. The effect of MePEG-PLA-CS nanoparticles on human liver cancer cells (HepG2) and normal liver cells (L-02) was studied by MTT method. PLA-CS carrying pEGFP-C1 (green fluorescent protein particle) pDNA as the reporter gene transfected COS7 cells to evaluate the transfection efficiency in vitro, in which commercial liposomes (Lipofectamine2000, Invitrogen) were used as positive controls and naked DNA as negative control. Further, the BALB / C tumor mouse model was constructed with MePEG-PLA-CS nanoparticles carrying the report plasmid green. Color fluorescence protein (EGFP) is used to study the ability of gene transfection in vivo.
Results: through the orthogonal experiment, we obtained the optimal conditions for the preparation of MePEG-PLA-CS NP. AFM and particle size analyzer proved that the nanoparticles were smooth and complete, dispersed well, and the surface potential was positive. The DNA binding efficiency (DNA loading efficiency) of the nanoscale carrier transport system (DNA loading efficiency) was 91% + 4.5%.MePEG-PLA-CS nanometers. It is also proved that the MePEG-PLA-CS nanoparticles / DNA complexes can protect the carrier DNA from nuclease degradation. This study found that MePEG-PLA-CS nanoparticles were not cytotoxic to normal liver cells (L-02) in a certain dose range, only in high concentration. The results showed that MePEG-PLA-CS nanoparticles were not cytotoxic to normal liver cells (L-02) in a certain dose range. In vitro gene transport, the nanoparticles can effectively transport EGFP (green fluorescent protein) to report that the gene expression plasmid entered COS7 cells, and its transport efficiency is 40%, stronger than the transfection efficiency of liposomes under the same condition. In the gene transfection experiment, the MePEG-PLA-CS nanoparticles are located at the nanoparticles. A group of nude mice has the highest expression of EGFP gene in tumor tissue, which confirms that MePEG-PLA-CS nanoparticles can be retained in the circulatory system for a long time without being captured by the reticuloendothelial system with the help of PEG, and are enriched in the tumor tissue through the effect of EPR (selective retention effect), and have the ability to target the tumor targeting the tumor.
Conclusion: using the dialysis filtration method and optimizing the technological conditions, the particles with small size and uniform distribution of MePEG-PLA-CS nanoparticles were successfully prepared. The nanoparticles were charged with positive charge and then combined with plasmid DNA to assemble the MePEG-PLA-CS nanoparticle gene transport system. The transfer system was confirmed through the transfection of the body and the body and the protection of DNA. The DNA is protected from the degradation of nuclease, and is a low toxic and efficient nanoscale gene carrier. Our research has laid a good scientific basis for the application of nanoparticles in the treatment of tumor gene therapy.

【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346

【引证文献】