聚阳离子纳米脂质载体的制备及穿膜机理的研究

发布时间：2018-07-05 05:38

本文选题：烷基化聚乙烯亚胺 + 三油酸甘油酯　；参考：《复旦大学》2008年博士论文

【摘要】：高效、安全的基因递送载体的研究是目前基因治疗研究的主要难点之一。非病毒基因递送载体由于安全、易组装等特点备受国内外学者的关注。本课题结合磷脂、聚乙烯亚胺(Polyethylenimine,PEI)以及油酸甘油酯的特点,制备了聚阳离子纳米脂质载体(Polycation nanostructured lipid carrier,PNLC),用于基因递送的研究。本文研究内容包括烷基化聚乙烯合成,PNLC的制备及组分的筛选;PNLC作为绿色荧光蛋白报告基因pEGFP-N2和虫荧光素酶报告基因pGL3-luc递送载体的研究;以SPC-A1细胞为模型,采用加入特异性抑制剂的方法,考察PNLC/DNA复合物的细胞内吞途径和转运机制;采用活细胞显微成像技术观察PNLC/DNA复合物进入细胞以及细胞内转运的过程。本文将溴化十四烷、溴化十六烷和溴化十八烷分别与聚乙烯亚胺(PEI600Da,PEI1200Da和PEI1800Da)反应,合成了27种烷基化聚乙烯亚胺,采用乳化溶剂蒸发法制备PNLC,并对制备工艺进行了优化,制备工艺优化为:超声功率200W,超声时间5min,分散介质为双蒸水。 PNLC由烷基化聚乙烯亚胺、油相和磷脂三种成分组成。烷基化聚乙烯亚胺为上述合成的27种不同的烷基修饰的聚乙烯亚胺;油相为油酸甘油酯、中链甘油三酯M812、三肉豆蔻酸甘油酯、三棕榈酸甘油酯和辛酸癸酸甘油酯M818;磷脂为卵磷脂EPC和二油酰磷脂酰乙醇胺DOPE。采用乳化溶剂蒸发法制备PNLC,测定其粒径和zeta电位,与pEGFP-N2质粒结合形成PNLC/DNA复合物,转染SPC-A1细胞,采用荧光分光光度法测定SPC-A1细胞中表达的绿色荧光蛋白的荧光强度,以SPC-A1细胞中表达的绿色荧光蛋白的荧光强度为指标,分别筛选PNLC的各项组成。选择油相为三油酸甘油酯、磷脂为卵磷脂EPC,考察烷基化聚乙烯亚胺对PNLC基因转染效率的影响。结果表明PNLC中烷基化聚乙烯亚胺为十六烷基化聚乙烯亚胺时,与三油酸甘油酯和卵磷脂组成的PNLC转染效率较高,而且当烷基化聚乙烯亚胺为十六烷基化PEI1200(1:6)即P_(12)C_(16)(1:6)时,PNLC在SPC-A1细胞中转染pEGFP-N2后,细胞中表达的绿色荧光蛋白的荧光强度与阳离子脂质体Lipofectamine~(TM)2000相当。因此,选择PNLC处方中烷基化聚乙烯亚胺为P_(12)C_(16)(1:6)。选择烷基化聚乙烯亚胺为P_(12)C_(16)(1:6),磷脂为卵磷脂EPC,考察油相对PNLC基因转染的影响。结果表明当油相为油酸甘油酯和中链甘油三酯M812时,制备的PNLC在SPC-A1细胞中转染pEGFP-N2质粒后,细胞中表达的绿色荧光蛋白的荧光强度较高。因此,选择PNLC的油相为中链甘油三酯M812和油酸甘油酯(三油酸甘油酯、二油酸甘油酯和单油酸甘油酯)。选择烷基化聚乙烯亚胺为P_(12)C_(16)(1:6),油相为中链甘油三酯M812、三油酸甘油酯、二油酸甘油酯和单油酸甘油酯,考察磷脂的种类(卵磷脂EPC或者二油酰磷脂酰乙醇胺DOPE)以及油相/磷脂的比值对PNLC基因转染的影响。结果表明当油相成分为油酸甘油酯时,PNLC在SPC-A1细胞中转染质粒pEGFP-N2后,细胞中表达的绿色荧光蛋白的荧光强度高于阳离子脂质体Lipofectamine~(TM)2000。PNLC处方中加入油酸甘油酯后,细胞中表达的绿色荧光蛋白的荧光强度提高。PNLC处方优化结果如下:在由P_(12)C_(16)(1:6)、油酸甘油酯和卵磷脂EPC组成的PNLC中,P_(12)C_(16)(1:6)与卵磷脂的摩尔比值为0.1,三油酸甘油酯与卵磷脂EPC的摩尔比值为1,二油酸甘油酯与卵磷脂EPC的质量比值为2,单油酸甘油酯与卵磷脂EPC的摩尔比值为6;在由P_(12)C_(16)(1:6)、油酸甘油酯和磷脂DOPE组成的PNLC中,P_(12)C_(16)(1:6)与磷脂DOPE的摩尔比值为0.1,三油酸甘油酯与磷脂DOPE的摩尔比值为0.8,二油酸甘油酯与磷脂DOPE的质量比值为0.75,单油酸甘油酯与磷脂DOPE的摩尔比值为1.5。上述六种组成PNLC在SPC-A1细胞和CHO细胞中转染质粒pEGFP-N2,流式细胞仪测定其转染效率,结果表明,在SPC-A1细胞和CHO细胞中,由P_(12)C_(16)(1:6)、三油酸甘油酯或者二油酸甘油酯、卵磷脂组成的PNLC转染效率与阳离子脂质体Lipofectamine~(TM)2000相当,而由P_(12)C_(16)(1:6)、单油酸甘油酯和卵磷脂组成的PNLC转染效率显著高于阳离子脂质体Lipofectamine~(TM)2000;由P_(12)C_(16)(1:6)、油酸甘油酯和磷脂DOPE组成的PNLC转染效率显著高于阳离子脂质体Lipofectamine~(TM)2000。PNLC中磷脂为DOPE时的基因转染效率较高。在含有10%血清的条件下,PNLC仍具有较高的基因转染效率。选择由P_(12)C_(16)(1:6)、三油酸甘油酯、DOPE组成的PNLC(PTD),由P_(12)C_(16)(1:6)、二油酸甘油酯和DOPE组成的PNLC(PDD),由P_(12)C_(16)(1:6)、单油酸甘油酯和卵磷脂EPC组成的PNLC(PME),在SPC-A1细胞和CHO细胞中转染虫荧光素酶报告基因pGI3-luc,结果表明在SPC-A1细胞中,三种组成的PNLC的转染效率显著高于阳离子脂质体Lipofectamine~(TM)2000,而且在含有10%血清的条件下具有较高的转染效率;在CHO细胞中,PTD和PDD组成的PNLC的转染效率显著高于阳离子脂质体Lipofectamine~(TM)2000,而且在含有10%血清的条件下,均具有较高的转染效率,但是PME组成的PNLC的转染效率非常低。选择PTD、PDD和PME组成的PNLC,以SPC-A1细胞为模型,采用加入特异性抑制剂的方法,考察PNLC/DNA复合物进入细胞的内吞途径以及细胞内的转运机制。分别加入网格蛋白内吞途径抑制剂氯丙嗪和陷穴小泡内吞途径Filipin,测定PNLC/DNA复合物的内吞情况以及虫荧光素酶报告基因在细胞内的表达情况。结果表明在SPC-A1细胞中,加入氯丙嗪后,PNLC/DNA复合物的内吞降低,细胞中表达的luciferase活性降低了近90%,而加入Filipin后,PNLC/DNA复合物的内吞和细胞中表达的luciferase的活性均没有显著性的变化。因此推断PNLC/DNA复合物通过网格蛋白介导的内吞途径进入细胞。为了证明这一推断,分别对PNLC/DNA复合物和细胞内溶酶体用荧光探针进行标记,用激光共聚焦显微镜观察发现PNLC/DNA复合物进入细胞后与溶酶体的重合,因此,PNLC/DNA复合物主要是通过网格蛋白介导的内吞途径进入细胞。细胞内微管和摩托蛋白对细胞内的膜泡转运具有非常重要的作用,加入微管解聚剂nocodazole和微管稳定剂paclitaxel,动力蛋白抑制剂原钒酸钠(SOV)和驱动蛋白Eg5抑制剂monastrol,测定SPC-A1细胞内表达的luciferase的活性,考察微管和摩托蛋白对于PNLC/DNA复合物在SPC-A1细胞内转运的影响。结果表明SPC-A1细胞中加入微管解聚剂nocodazole和微管稳定剂paclitaxel后,细胞中几乎没有luciferase表达;加入原钒酸钠后细胞中的luciferase的活性提高了50%,加入monastrol后PTD和PDD组成的PNLC转染后luciferase活性降低,而PME组成的PNLC转染后luciferase活性没有显著性变化,同时加入原钒酸钠和monastrol后,细胞内表达的luciferase活性均发生不同程度的降低。这些结果表明,微管和摩托蛋白对于PNLC/DNA复合物在细胞内的转运具有非常重要的影响。为了进一步考察PNLC/DNA复合物进入细胞的过程以及细胞内的转运情况,选择PME组成的PNLC,PNLC用绿色荧光探针FITC标记,质粒DNA用红色荧光探针TM-Rhodamine进行标记,用激光共聚焦显微镜跟踪PNLC/DNA复合物进入细胞以及在细胞内转运的过程。观察结果表明PNLC能够压缩质粒DNA形成PNLC/DNA复合物,内吞进入细胞后,通过早期内吞泡-晚期内吞泡-溶酶体的路径转运到细胞核核周围区域,烷基化聚乙烯亚胺和质粒DNA均可转运进入细胞核。
[Abstract]:The research of efficient and safe delivery vector for gene delivery is one of the main difficulties in the research of gene therapy. The non viral gene delivery carrier has attracted much attention from domestic and foreign scholars because of its safety and easy assembly. This subject has prepared polycationic nanoscale with the characteristics of phospholipid, Polyethylenimine, PEI and oleate. Polycation nanostructured lipid carrier (PNLC) is used for the study of gene delivery.
This article includes the synthesis of alkylated polyethylene, the preparation of PNLC and the screening of components, PNLC as the green fluorescent protein reporter gene pEGFP-N2 and the pGL3-luc delivery carrier of the luciferase reporter gene, and the method of adding specific inhibitors to SPC-A1 cells to investigate the endocytosis pathway of the PNLC/DNA complex. And the transport mechanism. The PNLC/DNA complex was introduced into the cells and the intracellular transport process was observed by living cell microscopy.
In this paper, brominated fourteen alkane, brominated sixteen alkane and brominated eighteen alkane were reacted with polyethyleneimine (PEI600Da, PEI1200Da and PEI1800Da) to synthesize 27 kinds of alkylated polyethyleneimine, and PNLC was prepared by emulsion solvent evaporation method. The preparation process was optimized. The preparation process was optimized by ultrasonic power 200W, ultrasonic time 5min, dispersion mediator. The quality is double steamed water.
PNLC consists of three kinds of alkylated polyethyleneimine, oil phase and phospholipid. Alkylated polyethyleneimine is the 27 different alkyl modified polyethyleneimine synthesized above; oil phase is oleic acid glyceride, medium chain triglyceride M812, three myrisate glyceride, three palmitate glyceride and octanoic acid glyceride M818; phosphatidylcholine is phosphatidylcholine EPC And two oil acyl phosphatidyl ethanolamine DOPE. was prepared by emulsion solvent evaporation method to prepare PNLC, determine its particle size and zeta potential, combine with pEGFP-N2 plasmid to form PNLC/DNA complex, transfect SPC-A1 cells, and use fluorescence spectrophotometry to determine the fluorescence intensity of green fluorescent protein expressed in SPC-A1 cells, and the green fluorescence expressed in SPC-A1 cells. The fluorescence intensity of protein was used as an index to screen the components of PNLC.
The effect of alkylated polyethylenimide on the transfection efficiency of PNLC gene was investigated by selecting oil as three oleate and phosphatidylcholine EPC. The results showed that when alkylated polyethyleneimine was sixteen alkylated polyethyleneimine in PNLC, the transfection efficiency of PNLC with three oleate and lecithin was higher than that of alkylated polyethyleneimine. When sixteen alkylated PEI1200 (1:6), P_ (12) C_ (16) (1:6), PNLC was transfected with pEGFP-N2 in SPC-A1 cells, the fluorescence intensity of the green fluorescent protein expressed in the cells was equivalent to the cationic liposome Lipofectamine~ (TM) 2000. Therefore, the alkylated polyethyleneimine was selected as P_ (12) (16) in the PNLC prescription.
Alkylated polyethyleneimine was selected as P_ (12) C_ (16) (1:6) and phospholipid as lecithin EPC. The effect of oil on the transfection of PNLC gene was investigated. The results showed that when the oil phase was oleate and medium chain triglyceride M812, the PNLC in SPC-A1 cells was transfected with pEGFP-N2 plasmids, and the fluorescence intensity of the green fluorescent protein expressed in the cells was higher. The oil phase of PNLC was selected as medium chain triglyceride M812 and glyceryl oleate (three glyceryl oleate, two oleic acid glyceride and monoglyceride).
Alkylated polyethyleneimine was selected as P_ (12) C_ (16) (1:6), oil phase was triglyceride M812, three oleate, two oleate and monoglyceride, and the effects of phosphatidylcholine (phosphatidylcholine EPC or two oleoyl ethanolamine DOPE) and oil phase / phospholipid ratio on PNLC gene transfection were investigated. The fluorescence intensity of the green fluorescent protein expressed in the cell was higher than that of the cationic liposome Lipofectamine~ (TM) 2000.PNLC. The fluorescence intensity of the green fluorescent protein expressed in the cell was improved by the Lipofectamine~ (TM) 2000.PNLC formulation. The fluorescence intensity of the green fluorescent protein expressed in the cell was improved by the.PNLC formulation. The results were as follows: in P_, the results were as follows: in P_, the results of the fluorescence intensity of the green fluorescent protein expressed in the cells were optimized as follows: in P_, the results were as follows: in P_, the fluorescence intensity of the green fluorescent protein expressed in the cells was as follows: in P_ (12) C_ (16) (16) (1:6), glycerol glyceride and lecithin EPC in PNLC, P_ (12) C_ (16) (1:6) and lecithin ratio 0.1, three olein glyceride and lecithin EPC, 1, two olein glyceride and phosphatidylcholine EPC ratio is 2, the molar ratio of glyceryl glyceride and lecithin EPC is 6; in P_ (12) C_ (16), The molar ratio of P_ (12) C_ (16) (12) C_ (16) (1:6) to phospholipid DOPE is 0.1, the ratio of three oleate to phospholipid DOPE is 0.8, the ratio of two oleate glyceride to phospholipid DOPE is 0.75, and the molar ratio of glycerol to phospholipid DOPE is 1.5. above SPC-A1 cells and six components PNLC. The transfection efficiency of O cells was detected by flow cytometry, and the transfection efficiency was measured by flow cytometry. The results showed that in SPC-A1 cells and CHO cells, P_ (12) C_ (16) (1:6), three oleate or two oleate, the PNLC transfection efficiency of lecithin was equivalent to that of cationic liposome Lipofectamine~ (TM) 2000, while P_ (12) C_ (16), monoglyceride PNLC transfection efficiency of ester and lecithin was significantly higher than that of cationic liposome Lipofectamine~ (TM) 2000; P_ (12) C_ (16) (1:6), oleate glyceride and phospholipid DOPE were significantly higher transfection efficiency than cationic liposome Lipofectamine~ (TM) 2000.PNLC, the gene transfection efficiency was higher when phosphor lipid was DOPE, which contained 10% serum conditions. At the same time, PNLC still has high gene transfection efficiency.
P_ (12) C_ (16) (16), three oleate, DOPE, PNLC (PTD), PNLC (PDD) consisting of P_ (12) C_ (16) (1:6), two oleate and DOPE, was composed of 12 (16), glycerol monoglyceride and lecithin. In 1 cells, the transfection efficiency of three components of PNLC was significantly higher than that of cationic liposome Lipofectamine~ (TM) 2000, and had higher transfection efficiency under the conditions of 10% serum. In CHO cells, the transfection efficiency of PNLC, which was composed of PTD and PDD, was significantly higher than that of cationic liposome Lipofectamine~ (TM) 2000, and in the 10% serum containing strips. The transfection efficiency is high, but the transfection efficiency of PNLC composed of PME is very low.
The PNLC, composed of PTD, PDD and PME, was used as a model of SPC-A1 cells. The endocytosis pathway of PNLC/DNA complex into cells and the mechanism of intracellular transport were investigated by adding specific inhibitors. The internal endocytosis pathway, chlorpromazine and trapping vesicle endocytic pathway Filipin, were added to the PNLC/DNA complex. Endocytosis and the expression of the luciferase reporter gene in the cells showed that the endocytosis of PNLC/DNA complex was reduced after adding chlorpromazine in SPC-A1 cells, and the activity of luciferase in the cells was reduced by nearly 90%. After adding Filipin, the activity of the endocytosis of PNLC/DNA complex and the expression of luciferase in the cells had not been found. Significant changes. Therefore, it is inferred that the PNLC/DNA complex enters cells through the endocytic pathway mediated by the gridin. In order to prove this inference, the PNLC/DNA complex and the intracellular lysosomes are labeled with fluorescence probes, and the coincidence of the PNLC/DNA complex to the lysosome after the entry of the complex is observed by laser confocal microscopy. The PNLC/DNA complex mainly enters the cell through the clathrin mediated endocytosis pathway.
Intracellular microtubules and motor proteins have a very important role in the transport of membrane vesicles in cells, including microtubule depolymerization agent nocodazole and microtubule stabilizer paclitaxel, SOV and Eg5 inhibitor Monastrol, the activity of luciferase in SPC-A1 cells, microtubules and MOS The effect on the transport of PNLC/DNA complex in SPC-A1 cells showed that there was almost no luciferase expression in the cells after the addition of microtubule depolymerization agent nocodazole and microtubule stabilizer paclitaxel, and the activity of luciferase in the cells after adding sodium vanadate was increased by 50%. After Monastrol, PTD and PDD made up of PNLC transfection after Monastrol. The activity of luciferase was reduced, and the activity of luciferase was not significantly changed after the transfection of PME, and the luciferase activity expressed in cells was reduced in varying degrees after adding sodium vanadate and Monastrol. These results showed that microtubules and motorproteins were very important for the transport of PNLC/ DNA complex in cells. Influence.
In order to further investigate the process of PNLC/DNA complex entering cells and the transport of cells, the PNLC of PME was selected, the PNLC was labeled with green fluorescent probe FITC, the plasmid DNA was marked with the red fluorescent probe TM-Rhodamine, and the laser confocal microscope was used to track the entry of PNLC/DNA complex and the process of transport in the cell. The results show that PNLC can compress plasmids DNA to form PNLC/DNA complex, and after endocytosis enters the cell, it can be transported to the nucleus around the nucleus through the pathway of the early endocytic vesicle and late endocytic vesicle lysosome, and the alkylated polyethyleneimine and plasmid DNA can be transported into the nucleus.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2008
【分类号】：R346

【共引文献】