多功能脂质体基因载体的构建及评价

发布时间：2018-10-20 16:11

【摘要】：多功能脂质体作为基因载体,在基因治疗中的应用越来越受到重视,展现出了广阔的应用前景。本文以叶酸-聚乙二醇-胆固醇半琥珀酸酯(F-PEG-CHEMS)和硬脂酰八聚精氨酸(R8)作为功能成分,构建多功能脂质体,并把它作为ASODN和质粒DNA的载体,考察其理化性质和细胞的摄取行为与转染效果,探索其作为基因药物细胞内给药系统的可行性。首先,本文在调研有关文献基础上,设计出了胆固醇半琥珀酸酯(CHEMS)、聚乙二醇单甲醚-胆固醇半琥珀酸酯(MPEG-CHEMS)和F-PEG-CHEMS的合成路线。CHEMS是以胆固醇和丁二酸酐为原料通过酯化反应制备；MPEG-CHEMS是以聚乙二醇单甲醚(MPEG)和CHEMS为原料通过酯化反应制备；F-PEG-CHEMS的合成分三步完成：首先叶酸与聚乙二醇二胺(NH2-PEG-NH2)反应制备F-PEG2000-NH2,然后胆固醇半琥珀酸酯(CHEMS)与N-羟基琥珀酰亚胺(NHS)反应制备NHS-CHEMS,最后F-PEG-NH2与NHS-CHEMS反应,得到F-PEG-CHEMS。合成并纯化的产物经TLC, IR,.1H-HMR质谱和1H-HMR谱分析,确证为目标产物。合成并纯化了的脂质体膜材MPEG-CHEMS与F-PEG-CHEMS,为多功能脂质体的制备奠定了基础。其次,用薄膜分散法制备普通脂质体,并把R8水溶液与普通脂质体在45℃孵育1h,制备R8修饰的脂质体(R8-Lip),同时对其进行理化性质和细胞水平的评价。实验结果表明,普通脂质体经R8修饰后,粒径和分布基本不变,而zeta电位由-10.24mV变为+38.69mV。当R8-Lip/ASODN复合物中+／-超过4：1时,ASODN已与R8-Lip完全结合成带正电的复合物；当R8-Lip中R8的浓度小于10μg/mL时,细胞存活率大于75%;R8-Lip对ASODN细胞摄取的促进作用与R8的浓度有关,当R8的质量浓度由0.3μg/mL增加到9.6μg/mL(+／-由1：2增加到16：1),细胞内荧光强度逐渐增加；当R8-Lip/ASODN复合物中+／-=16：1时,R8对ASODN摄取的促进作用强于市售的转染试剂Lipofectamine2000。在R8-Lip表面PEG化修饰后,促进细胞摄取ASODN的效果很差。为了进一步提高细胞摄取效果,在PEG末端接上叶酸配体,构建了多功能脂质体R8-Lip-F,并对其进行理化性质和细胞水平的评价。实验结果表明,该脂质体的形态规整,几乎全部呈球形。R8-Lip-F/ASODN在+／-为16：1时复合物完全被阻滞在上样口。R8-Lip-F/ASODN复合物在6h内的细胞摄取量和阳性率随时间的增加而增加。R8-Lip-F/ASODN复合物通过内吞途径入胞,且具有能量依赖性。其入胞机制主要是网格蛋白介导的内吞和巨胞饮作用,并且细胞摄取量会因叶酸的竞争性抑制而降低。ASODN入胞后大部分分布在胞浆中,细胞核中几乎没有。与市售的转染试剂Lipofectamine2000相比,R8-Lip-F对ASODN摄取的促进作用要强一些,但其对质粒DNA的转染几乎无促进作用,转染效果远差于市售的转染试剂Lipofectamine2000。最后,以DOTAP脂质体为基础,并在其中引入F-PEG-CHEMS与R8,构建多功能阳离子脂质体R8-DOTAP-F,同时对其进行理化性质和细胞水平的评价。实验结果表明,R8-DOTAP-F的形态规整,几乎全部呈球形。在R8-DOTAP-F复合物中DOTAP与pDNA或ASODN质量比为30(正负电荷比或N/P约为13：1)时,pDNA和ASODN被结合完全。R8-DOTAP-F/ASODN复合物在6h内的细胞摄取量和阳性率随时间的增加而增加。对脂质体进行叶酸配体修饰,可以明显提高脂质体复合物的细胞摄取与转染效果,并且,对脂质体进行R8修饰可以进一步提高细胞转染与摄取效果。R8-Lip-F/ASODN复合物通过内吞途径入胞,且具有能量依赖性。其入胞机制主要是网格蛋白介导的内吞和巨胞饮作用,并且细胞摄取量会因叶酸的竞争性抑制而降低。入胞后ASODN大部分分布在细胞核中,溶酶体中几乎没有。与市售的转染试剂Lipofectamine2000相比,R8-DOTAP-F对ASODN摄取的促进作用要强很多,但其对质粒DNA转染的促进作用要略差一些。
[Abstract]:The multifunctional liposome is used as a gene vector, and the application of the multifunctional liposome in gene therapy is more and more important, and has a wide application prospect. A multifunctional liposome was constructed by taking folic acid-polyethylene glycol-cholesterol semi-succinate (F-PEG-CHEMS) and stearin 8-poly arginine (R8) as functional components, and taking it as carrier of ASODN and plasmid DNA. To explore the feasibility of the drug delivery system as a gene drug cell. Firstly, the synthesis of cholesterol semi-succinate (CHEMS), polyethylene glycol monomethyl ether-cholesterol semi-succinate (MPEG-CHEMS) and F-PEG-CHEMS was designed based on the literature. The synthesis of F-PEG-CHEMS is carried out by reaction of folic acid with polyethylene glycol diamine (NH2-PEG-NH2). H2 and then reacting cholesterol semi-succinic acid ester (CHEMS) with N-hydroxyamber to prepare the F-CHEMS, and finally reacting the F-PEG-NH2 with the NHS-CHEMS to obtain the F-PEG-NH2. MS. The synthesized and purified products were identified as targets by TLC, IR,. 1H-HMR mass spectrometry and 1H-HMR spectroscopy The synthesized and purified liposome membrane material MPEG-CHEMS and F-PEG-CHEMS have laid the foundation for the preparation of multifunctional liposomes. secondly, preparing the common liposome by a thin film dispersion method and incubating the R8 aqueous solution with the common liposome at 45 DEG C for 1h to prepare R8-modified liposome (R8-Lip), The experimental results show that the particle size and distribution of the liposomes are basically unchanged after R8 is modified, and the zeta potential is changed from -10. 24mV to + 38.. 69mV. When +/-exceeds 4: 1 in R8-Lip/ ASODN complex, ASODN has been fully integrated with R8-Lip to positively charged complex; when R8-Lip is less than 10 & mu; g/ mL, cell survival is greater than 75%; R8-Lip contributes to the uptake of ASODN cells and R8 The concentration of R8 is related to the increase of fluorescence intensity in cells when the mass concentration of R8 is increased from 0.3. m u.g/ mL to 9. 6. m u.g/ mL (+/-increased from 1: 2 to 16: 1); when +/-= 16: 1 in R8-Lip/ ASODN complex, R8 promotes the uptake of ASODN stronger than the commercially available transfection reagent, Lipofectin e2000. After PEGylation modification on R8-Lip surface, promote cell uptake AS In order to further improve the effect of cell uptake, a multifunctional liposome R8-Lip-F was constructed and its physicochemical properties were studied in order to further improve the effect of cell uptake. The results show that the morphology of the liposome is regular. Almost all spherical. R8-Lip-F/ ASODN is completed at +/-16: 1 The cellular uptake and positive rate of the R8-Lip-F/ ASODN complex in 6h increased with the increase in time increases. R8-Lip-F/ ASODN complexes are introduced into the cell by an endocytic pathway, It has an energy-dependent mechanism. Its intracellular mechanism is mainly protein-mediated endocytic and giant cell drinking, and the cellular uptake is due to folic acid. competitive inhibition decreased. The majority of ASODN was distributed in cytoplasm after ASODN was introduced into the cytoplasm. The effect of R8-Lip-F on the uptake of ASODN was lower than that of the transfection reagent which was sold in the city. However, it had little effect on the transfection of plasmid DNA. The transfection efficiency was far from the transfection reagent of the market. mine2000, finally, using DOTAP liposome as the basis, introducing F-PEG-CHEMS and R8 in it, constructing multifunctional cationic liposome R8-DOTAP-F, The results show that R8-DOTAP-F In the R8-DOTAP-F complex, pDNA is present in the R8-DOTAP-F complex with a mass ratio of DOTAP to pDNA or ASODN of 30 (positive/ negative charge ratio or N/ P of about 13: 1). and ASODN are combined completely. R8-DOTAP-F/ ASODN complex has cell intake and yang in 6h According to the modification of the folic acid ligand on the liposome, the cell uptake and transfection effect of the liposome complex can be obviously improved, and the R8 modification on the liposome can further Enhancement of cell transfection and uptake results. R8-Lip-F/ ASODN complexes pass endocytic pathways The intracellular mechanism is primarily a protein-mediated endocytic and macrocellular uptake, and the cellular uptake is due to The competitive inhibition of folic acid decreases. The majority of ASODN after entering the cell is distributed in cells. Almost none of the lysosomes in the nucleus. Compared with the transfection reagent which was sold in the market, R8-DOTAP-F promoted the uptake of ASODN.
【学位授予单位】：浙江工业大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R943

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