半乳糖衍生物阳离子脂质体基因载体的合成与性能研究

发布时间：2019-02-11 17:59

【摘要】：基因治疗需要开发高效、安全的载体系统,用于运载DNA或siRNA至目标组织。阳离子脂质体作为一类重要的非病毒载体,具有容易制备,重现性好,无免疫原性,能与大分子DNA形成稳定的复合物等优势。本文以半乳糖为原料,合成了两类阳离子脂质体,第一类经过全乙酰化、脱1-O-乙酰基、三氯乙酰亚胺酯化、糖苷化、叠氮化、脱乙酰基保护、3,4-O-异丙叉基保护、醚化、脱异丙叉基、还原胺化和季铵盐化反应,合成不同长度疏水链连在糖环氧原子上的半乳糖衍生物阳离子脂质体:di-C12-Gal-TMA,di-C14-Gal-TMA,di-C16-Gal-TMA,di-C18-Gal-TMA;第二类经过全乙酰化、脱1-O-乙酰基、三氯乙酰亚胺酯化、糖苷化、叠氮化、脱乙酰基保护、还原氨化、叔胺化、季铵盐化反应,合成不同长度疏水链连在季铵盐头部氮原子上的半乳糖衍生物阳离子脂质体:Gal-DiC12MA,Gal-DiC14MA,Gal-DiC16MA,Gal-DiC18MA。采用马尔文激光粒度仪测定阳离子脂质体及其DNA复合物的Zeta电位、平均粒径、PDI。结果显示:阳离子脂质体的Zeta电位在50-70 mv之间;平均粒径在130-245 nm之间,分布均匀;脂质体/DNA复合物Zeta电位随着N/P的增大而增大,第一类脂质体复合物在N/P大于4时达到正值,第二类脂质体复合物在N/P为2时就已为正值;除了di-C18-Gal-TMA/DNA,Gal-DiC18MA/DNA在低N/P时,平均粒径为300-450 nm,其余复合物的平均粒径在100-250 nm之间。用原子力显微镜表征阳离子脂质体及其DNA复合物的表面形貌,显示所有阳离子脂质体及其DNA复合物都呈球形,分散均匀。通过凝胶阻滞实验探究脂质体与DNA的结合能力,随着N/P的增大DNA在凝胶中的阻滞作用增强,除了di-C18-Gal-TMA,其余脂质体在N/P达到2时DNA已被结合完全。以Lipo2000作阳性对照,分别检测阳离子脂质体携带DNA在PC-3,HEK293,HepG2,Mat,Hela,SW480,A375,7721细胞内的表达效果,以及阳离子脂质体携带siRNA在PC-3、Mat细胞中的基因沉默效果。结果表明:di-C16-Gal-TMA/DNA在PC-3,Mat,Hela细胞中有较好的表达效果;而Gal-DiC16MA/DNA在HEK293,HepG2细胞中有较好的表达效果;di-C16-Gal-TMA/siRNA和Gal-DiC16MA/siRNA在PC-3,Mat细胞中都有较好的基因沉默效果。同时细胞毒性实验表明di-C16-Gal-TMA,Gal-DiC16MA在PC-3,HEK293,Hep G2,Mat,Hela五种细胞中的细胞毒性都较低。因此值得对di-C16-Gal-TMA,Gal-DiC16MA做更进一步的研究。
[Abstract]:Gene therapy requires the development of efficient and safe vector systems for carrying DNA or siRNA to target tissues. As a kind of important non-viral carrier, cationic liposomes have the advantages of easy preparation, good reproducibility, no immunogenicity, and the ability to form stable complexes with macromolecular DNA. In this paper, two kinds of cationic liposomes were synthesized from galactose. The first kind of liposomes were completely acetylated, deacetylated, trichloroimide esterified, glycosylated, azide, deacetylated, and 34-Oisopropylidene protected. The cationic liposomes of galactose derivatives were synthesized by etherification, deisopropyl group, reductive amination and quaternary ammonium salinization. The cationic liposomes of galactose derivatives with different lengths of hydrophobic chain were synthesized on glycosyl epoxy atom: di-C12-Gal-TMA,di-C14-Gal-TMA,di-C16-Gal-TMA, Di-C18-Gal-TMA; The second type is completely acetylated, deacetylated, esterified with trichloroimide, glycoside, azide, deacetylation, reductive ammoniation, tertiary amination, quaternary ammonium salinization, Synthesis of galactose Derivatives Cationic Liposomes with different length hydrophobic chains attached to nitrogen Atom of Quaternary ammonium Salt head: Gal-DiC12MA,Gal-DiC14MA,Gal-DiC16MA,Gal-DiC18MA. Determination of Zeta potential, mean particle size, PDI. of cationic liposomes and their DNA complexes by Ma Erwen laser particle size analyzer The results showed that the Zeta potential of cationic liposomes ranged from 50 to 70 mv and the average particle size ranged from 130 to 245 nm. The Zeta potential of liposome / DNA complex increases with the increase of N / P, the first type of liposome complex reaches positive value when N / P is greater than 4, and the second type of liposome complex is positive when N / P is 2. With the exception of di-C18-Gal-TMA/DNA,Gal-DiC18MA/DNA at low N / P, the average particle size of the complexes ranged from 300-450 nm, to 100-250 nm. The surface morphology of cationic liposomes and their DNA complexes was characterized by atomic force microscopy. The results showed that all cationic liposomes and their DNA complexes were spherical and uniformly dispersed. The binding ability of liposome to DNA was investigated by gel blocking experiment. With the increase of N / P, the blocking effect of DNA in gel was enhanced, and the other liposomes, except di-C18-Gal-TMA, had been completely bound at 2 DNA at N / P level. The effect of cationic liposome carrying DNA in PC-3,HEK293,HepG2,Mat,Hela,SW480,A375,7721 cells and the gene silencing effect of cationic liposome carrying siRNA in PC-3,Mat cells were detected by using Lipo2000 as positive control. The results showed that the expression of di-C16-Gal-TMA/DNA in PC-3,Mat,Hela cells was better than that of Gal-DiC16MA/DNA in HEK293,HepG2 cells. Both di-C16-Gal-TMA/siRNA and Gal-DiC16MA/siRNA have good gene silencing effect in PC-3,Mat cells. At the same time, the cytotoxicity of di-C16-Gal-TMA,Gal-DiC16MA in PC-3,HEK293,Hep G _ (2) T _ (2) H _ (2) was lower. Therefore, it is worth doing further research on di-C16-Gal-TMA,Gal-DiC16MA.
【学位授予单位】：湖南师范大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q789

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