制备高效、安全树形高分子基因载体的策略

发布时间：2018-02-02 23:59

本文关键词： 树形高分子转染效率细胞毒性分子量氮磷比可逆化学交联氟化修饰策略　出处：《华东师范大学》2016年博士论文　论文类型：学位论文

【摘要】：树形高分子是一类人工合成的阳离子高分子,被广泛用作非病毒基因转染载体。这类高分子具有良好的单分散性、可控的尺寸和表面官能团、良好的水溶性及生物相容性。其表面的正电荷可结合核酸,内部三级胺具有质子缓冲效应,可促进转染复合物的内涵体逃逸。目前针对树形高分子作为基因载体已经有大量的研究工作,基于树形高分子的转染试剂SuperFect汶和PolyFect汶己实现商业化。然而,树形高分子作为基因转染载体在应用过程中存在着转染效率和细胞毒性之间的恶性关联：(1)随着树形高分子分子量的增加,其转染效率提高,但是其细胞毒性也会相应增大；(2)随着树形高分子与核酸复合物氮磷比的增加,其转染效率提高,但是其细胞毒性也会相应增大。因此,如何打破树形高分子基因转染过程中转染效率与细胞毒性的恶性关联是一个重要的科学问题。本文旨在解决树形高分子基因转染过程中的转染效率与细胞毒性恶性关联的难题,具体策略如下：(1)通过可逆化学交联的策略交联低分子量树形高分子,得到高效、安全的树形高分子基因载体。我们用含二硫键的双功能交联剂交联小分子量(分子量为3256 Da)的聚酰胺-胺树形高分子,得到可降解的纳米聚集体。研究表明该策略未影响树形高分子结合和保护核酸的能力。该策略制备的纳米聚集体具有强的细胞摄入能力和较高的基因转染效率。该纳米聚集体具有高于大分子量聚酰胺-胺树形高分子的转染效率,并与支化的聚乙烯亚胺(分子量为25 kDa)的转染效率相当。另外,该纳米聚集体具有良好的生物相容性,其细胞毒性远低于大分子量树形高分子和支化的聚乙烯亚胺的细胞毒性。我们进一步验证了二硫键的断裂在该纳米聚集体基因转染过程中的关键作用。因此通过可逆化学交联法我们实现了基于小分子量树形高分子的高效、低毒的基因转染。(2)通过氟化修饰策略制备高效、安全的树形高分子基因载体。我们通过简单的化学反应在聚酰胺-胺树形高分子表面修饰含氟脂肪链,得到一系列氟化修饰的树形高分子基因载体。这类载体含氟基团之间的亲氟效应极大地提高了载体与核酸复合物的稳定性,从而可在低氮磷比条件下形成稳定的复合物,低氮磷比条件的转染将产生低的细胞毒性。这类载体在低氮磷比条件下实现了高效、低毒的转染。例如七氟丁酸酐修饰的聚酰胺-胺树形高分子与绿色荧光蛋白基因在氮磷比为1.5：1时,在HEK293细胞可达到90%以上的转染效率；氮磷比为2：1时在HeLa细胞可达到90%以上的转染效率,即使在整个复合物系统呈电负性(氮磷比为0.5：1)时其转染效率仍高达50%。除了HEK293和HeLa细胞上,七氟丁酸酐修饰的聚酰胺-胺树形高分子在CHO、COS-7、MB231、A549、U2OS、 NIH3T3、MG63细胞上,在转染氮磷比在2：1左右均实现了高效、低毒的基因转染,其转染效率超过商业化的转染试剂Lipofectamine汶 2000等。因此,通过氟化修饰策略实现了树形高分子在较低氮磷比的转染,该策略制备的基因载体兼具高效、安全的特点。(3)以低分子量树形高分子和低氮磷比转染为出发点,利用氟化修饰策略制备高效、安全的树形高分子基因载体。聚丙烯亚胺树形高分子具有较小的分子量和较低的基因转染效率。我们在聚丙烯亚胺树形高分子表面修饰含氟脂肪链,得到一系列氟化修饰的聚丙烯亚胺树形高分子基因载体。这类含氟基因载体在低氮磷比条件可实现高效、低毒基因转染。例如将氟化修饰策略应用于小分子量的聚丙烯亚胺树形高分子(分子量为1687Da),得到的基因载体转染绿色荧光蛋白基因时,在氮磷比为1.6：1条件下可达到80%以上的转染效率。氟化修饰的第三、四、五代聚丙烯亚胺树形高分子基因载体的最佳转染氮磷比均在2：1以下,具有与商业化转染试剂Lipofectamine汶 2000、SuperFect汶、 PolyFect汶、jetPEI汶、 bPEI25K相当或者更优的转染效率。这类氟化修饰的基因载体在转染浓度下具有非常好的生物相容性。此外,该策略制备的树形高分子基因载体在3D多细胞球体上也可实现高效的基因转染。因此,氟化修饰策略实现了低分子量树形高分子在低氮磷条件下高效、低毒的基因转染。(4)利用氟化修饰方法制备高效、低毒的siRNA载体。我们分别在第五代聚酰胺-胺树形高分子表面修饰了不同种类的含氟化合物,合成了71个含氟功能基团修饰的树形高分子基因载体。我们筛选了这些氟化修饰载体的siRNA转染效率,其中10个含氟功能基团修饰的树形高分子可实现高效、低毒的基因沉默效率。有3个含氟基团修饰的树形高分子可在极低氮磷比(氮磷比为0.84：1、0.75：1和1.5：1)条件下达到80%以上的基因沉默效率,而未修饰的聚酰胺-胺树形高分子在最佳转染氮磷比为18：1的条件下只能实现10%以下的基因沉默效率。因此,通过氟化修饰的方法,我们得到了一系列基于树形高分子的基因载体,可以在低氮磷比条件下实现高效、低毒的siRNA转染。总而言之,本论文基于树形高分子作为基因转染载体的应用过程中存在的转染效率与细胞毒性的恶性关联,通过可逆化合交联法、氟化修饰等策略,制备了一系列高效、低毒的树形高分子基因(DNA和siRNA)载体。
[Abstract]:Dendrimer is a kind of synthetic cationic polymer, is widely used as a non viral gene vector. This kind of polymer has good dispersibility, size and surface functional groups of controllable, good water soluble property and biocompatibility. The surface of the positive charge can be combined with the nuclear acid, internal three amines with proton buffering effect of transfection complex can promote endosomal escape. The dendrimer as gene carrier has a lot of research work, the dendrimer transfection reagent SuperFect and PolyFect realized Wen Wen commercial based. However, dendrimer as gene carrier exists between malignant transfection efficiency and cytotoxicity in the application process in: (1) with increasing dendrimer molecular weight, improve the transfection efficiency, but its cytotoxicity will increase accordingly; (2) with tree shaped polymer With the nucleic acid complexes of nitrogen and phosphorus ratio increase, improve the transfection efficiency, but its cytotoxicity will increase accordingly. Therefore, how to break the vicious Association in the process of dendrimer gene transfection efficiency and cytotoxicity is an important scientific problem. This paper aims to solve the difficult problem of transfection efficiency and cytotoxicity associated with malignant tree shaped polymer in the gene transfection process, specific strategies are as follows: (1) the strategy of low molecular crosslinking reversible chemical crosslinking amount of dendrimer, efficient dendrimer gene carrier safety. We use bifunctional crosslinking agent molecule containing two sulfur bond amount (molecular weight of 3256 Da) the polyamideamine tree polymer, get biodegradable nano aggregates. Research showed that the strategy did not affect the dendrimer binding and protection of nucleic acids. The strategy for preparing nano aggregates having a strong cell The gene transfection efficiency of intake ability and higher. The nano structure is higher than that of the high molecular weight of PAMAM dendrimer transfection efficiency, and branched polyethyleneimine (molecular weight of 25 kDa) transfection efficiency. In addition, the nano aggregates have good biocompatibility, its cytotoxicity is far lower than that of large molecules the amount of dendrimer and branched polyethyleneimine of cytotoxicity. We further verified the key role of breaking two disulfide bonds in the nano aggregates in the gene transfection process. Therefore by reversible chemical crosslinking method we implemented based on small molecular weight dendrimers, gene transfection, lowtoxicity. (2) by fluoride modified strategy preparation of efficient and safe gene carrier dendrimers. Through simple chemical reactions in PAMAM dendrimer surface modification of fluorinated aliphatic chain, get a series of Dendrimer modified fluoride gene vector. These vectors containing fluorine groups between the parental effects greatly improves the stability of carrier and nucleic acid complexes, which can form stable complexes than under the condition of low nitrogen and phosphorus, with low nitrogen and phosphorus ratio will have low cytotoxicity. This kind of carrier in the low ratio of nitrogen and phosphorus under the conditions of achieving efficient, low toxicity. For example, transfected with green fluorescent protein amine dendrimer polyamide seven fluorine succinic anhydride modified genes in nitrogen and phosphorus ratio was 1.5:1, the HEK293 cells can reach more than 90% transfection efficiency; nitrogen and phosphorus ratio of 2:1 in HeLa cells can reach more than 90% the transfection efficiency, even in the whole complex system with electronegative (P = 0.5:1) when the transfection efficiency is as high as 50%. in addition to HEK293 and HeLa cells, polyamide seven fluorine succinic anhydride modified amine dendrimer in CHO, COS-7, MB231, A5 49, U2OS, NIH3T3, MG63 cells, the transfection ratio is about 2:1 in nitrogen and phosphorus are efficient, low toxicity gene transfection, the transfection efficiency of more than Lipofectamine Wen commercial transfection reagent 2000. Therefore, the fluoride modified strategy to achieve the dendrimer in the low transfection ratio of nitrogen and phosphorus, the gene carrier the preparation method has high efficiency, safety features. (3) with low molecular weight dendrimers and low ratio of nitrogen and phosphorus transfer as the starting point, the use of fluoride modification strategies for the preparation of highly efficient and safe gene carrier dendrimer. Molecular weight polypropylene imine dendrimers with small and low transfection efficiency. We modified fluorinated aliphatic chain in polypropylenimine dendrimer surface, polypropylenimine dendrimer has a series of fluoride modified gene carrier. This kind of fluorine containing gene carrier in low nitrogen and phosphorus ratio can achieve high efficiency and low toxicity. Gene transfection for example polypropylenimine dendrimer fluoride modification strategy is applied to small molecular weight (molecular weight 1687Da), green fluorescent protein gene transfection gene was obtained when the ratio of nitrogen to phosphorus in the conditions of 1.6:1 can reach more than 80%. The transfection efficiency of fluoride modified third, fourth, five generation polypropylenimine dendrimer gene the carrier of the best transfection ratio of nitrogen and phosphorus are below 2:1, with commercial transfection reagent Lipofectamine 2000 SuperFect PolyFect Wen, Wen, Wen Wen, jetPEI, the transfection efficiency of bPEI25K is equal to or better. This kind of gene carrier with fluorine modified excellent biocompatibility in transfection concentration. In addition, the strategy dendrimer gene carrier preparation in 3D multicellular spheroids can achieve high gene transfection efficiency. Therefore, fluoride modified strategy to achieve the low molecular weight dendrimers in low nitrogen and phosphorus Under the condition of high efficiency, low toxicity gene transfection. (4) the use of fluoride modified preparation method of high-efficiency, low toxicity siRNA vector. We were in the fifth generation of PAMAM dendrimer modified fluorine compounds containing different kinds of dendrimer gene carrier 71 fluorine modified functional groups were synthesized. We screened siRNA the transfection efficiency of these fluorinated dendrimer modified carrier, which 10 fluorinated functional groups of modified can realize the high efficiency, low toxicity gene silencing efficiency. There are 3 fluorinated dendrimer modified in low nitrogen phosphorus ratio (n / P ratio of 0.84:1,0.75:1 and 1.5:1) under the condition of more than 80% gene silencing efficiency however, non modified polyamide amine dendrimer in nitrogen and phosphorus than the optimal transfection condition of 18:1 was only 10% of the gene silencing efficiency. Therefore, through the method of fluoride modification, we have A series of dendrimers based on gene carrier, can be in low ratio of nitrogen and phosphorus under the conditions of efficient, low toxicity siRNA transfection. In short, this paper based on dendrimer as close the transfection efficiency and cytotoxicity of malignant process of gene vector in combination, through the combination of reversible cross-linking method, fluoride modification strategy the preparation of a series of high efficiency, low toxicity, dendrimer gene (DNA and siRNA) carrier.

【学位授予单位】：华东师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78

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