本文關(guān)鍵詞： 樹形高分子 轉(zhuǎn)染效率 細(xì)胞毒性 分子量 氮磷比 可逆化學(xué)交聯(lián) 氟化修飾策略　出處：《華東師范大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：樹形高分子是一類人工合成的陽離子高分子,被廣泛用作非病毒基因轉(zhuǎn)染載體。這類高分子具有良好的單分散性、可控的尺寸和表面官能團(tuán)、良好的水溶性及生物相容性。其表面的正電荷可結(jié)合核酸,內(nèi)部三級胺具有質(zhì)子緩沖效應(yīng),可促進(jìn)轉(zhuǎn)染復(fù)合物的內(nèi)涵體逃逸。目前針對樹形高分子作為基因載體已經(jīng)有大量的研究工作,基于樹形高分子的轉(zhuǎn)染試劑SuperFect汶和PolyFect汶己實(shí)現(xiàn)商業(yè)化。然而,樹形高分子作為基因轉(zhuǎn)染載體在應(yīng)用過程中存在著轉(zhuǎn)染效率和細(xì)胞毒性之間的惡性關(guān)聯(lián)：(1)隨著樹形高分子分子量的增加,其轉(zhuǎn)染效率提高,但是其細(xì)胞毒性也會相應(yīng)增大；(2)隨著樹形高分子與核酸復(fù)合物氮磷比的增加,其轉(zhuǎn)染效率提高,但是其細(xì)胞毒性也會相應(yīng)增大。因此,如何打破樹形高分子基因轉(zhuǎn)染過程中轉(zhuǎn)染效率與細(xì)胞毒性的惡性關(guān)聯(lián)是一個重要的科學(xué)問題。本文旨在解決樹形高分子基因轉(zhuǎn)染過程中的轉(zhuǎn)染效率與細(xì)胞毒性惡性關(guān)聯(lián)的難題,具體策略如下：(1)通過可逆化學(xué)交聯(lián)的策略交聯(lián)低分子量樹形高分子,得到高效、安全的樹形高分子基因載體。我們用含二硫鍵的雙功能交聯(lián)劑交聯(lián)小分子量(分子量為3256 Da)的聚酰胺-胺樹形高分子,得到可降解的納米聚集體。研究表明該策略未影響樹形高分子結(jié)合和保護(hù)核酸的能力。該策略制備的納米聚集體具有強(qiáng)的細(xì)胞攝入能力和較高的基因轉(zhuǎn)染效率。該納米聚集體具有高于大分子量聚酰胺-胺樹形高分子的轉(zhuǎn)染效率,并與支化的聚乙烯亞胺(分子量為25 kDa)的轉(zhuǎn)染效率相當(dāng)。另外,該納米聚集體具有良好的生物相容性,其細(xì)胞毒性遠(yuǎn)低于大分子量樹形高分子和支化的聚乙烯亞胺的細(xì)胞毒性。我們進(jìn)一步驗(yàn)證了二硫鍵的斷裂在該納米聚集體基因轉(zhuǎn)染過程中的關(guān)鍵作用。因此通過可逆化學(xué)交聯(lián)法我們實(shí)現(xiàn)了基于小分子量樹形高分子的高效、低毒的基因轉(zhuǎn)染。(2)通過氟化修飾策略制備高效、安全的樹形高分子基因載體。我們通過簡單的化學(xué)反應(yīng)在聚酰胺-胺樹形高分子表面修飾含氟脂肪鏈,得到一系列氟化修飾的樹形高分子基因載體。這類載體含氟基團(tuán)之間的親氟效應(yīng)極大地提高了載體與核酸復(fù)合物的穩(wěn)定性,從而可在低氮磷比條件下形成穩(wěn)定的復(fù)合物,低氮磷比條件的轉(zhuǎn)染將產(chǎn)生低的細(xì)胞毒性。這類載體在低氮磷比條件下實(shí)現(xiàn)了高效、低毒的轉(zhuǎn)染。例如七氟丁酸酐修飾的聚酰胺-胺樹形高分子與綠色熒光蛋白基因在氮磷比為1.5：1時,在HEK293細(xì)胞可達(dá)到90%以上的轉(zhuǎn)染效率；氮磷比為2：1時在HeLa細(xì)胞可達(dá)到90%以上的轉(zhuǎn)染效率,即使在整個復(fù)合物系統(tǒng)呈電負(fù)性(氮磷比為0.5：1)時其轉(zhuǎn)染效率仍高達(dá)50%。除了HEK293和HeLa細(xì)胞上,七氟丁酸酐修飾的聚酰胺-胺樹形高分子在CHO、COS-7、MB231、A549、U2OS、 NIH3T3、MG63細(xì)胞上,在轉(zhuǎn)染氮磷比在2：1左右均實(shí)現(xiàn)了高效、低毒的基因轉(zhuǎn)染,其轉(zhuǎn)染效率超過商業(yè)化的轉(zhuǎn)染試劑Lipofectamine汶 2000等。因此,通過氟化修飾策略實(shí)現(xiàn)了樹形高分子在較低氮磷比的轉(zhuǎn)染,該策略制備的基因載體兼具高效、安全的特點(diǎn)。(3)以低分子量樹形高分子和低氮磷比轉(zhuǎn)染為出發(fā)點(diǎn),利用氟化修飾策略制備高效、安全的樹形高分子基因載體。聚丙烯亞胺樹形高分子具有較小的分子量和較低的基因轉(zhuǎn)染效率。我們在聚丙烯亞胺樹形高分子表面修飾含氟脂肪鏈,得到一系列氟化修飾的聚丙烯亞胺樹形高分子基因載體。這類含氟基因載體在低氮磷比條件可實(shí)現(xiàn)高效、低毒基因轉(zhuǎn)染。例如將氟化修飾策略應(yīng)用于小分子量的聚丙烯亞胺樹形高分子(分子量為1687Da),得到的基因載體轉(zhuǎn)染綠色熒光蛋白基因時,在氮磷比為1.6：1條件下可達(dá)到80%以上的轉(zhuǎn)染效率。氟化修飾的第三、四、五代聚丙烯亞胺樹形高分子基因載體的最佳轉(zhuǎn)染氮磷比均在2：1以下,具有與商業(yè)化轉(zhuǎn)染試劑Lipofectamine汶 2000、SuperFect汶、 PolyFect汶、jetPEI汶、 bPEI25K相當(dāng)或者更優(yōu)的轉(zhuǎn)染效率。這類氟化修飾的基因載體在轉(zhuǎn)染濃度下具有非常好的生物相容性。此外,該策略制備的樹形高分子基因載體在3D多細(xì)胞球體上也可實(shí)現(xiàn)高效的基因轉(zhuǎn)染。因此,氟化修飾策略實(shí)現(xiàn)了低分子量樹形高分子在低氮磷條件下高效、低毒的基因轉(zhuǎn)染。(4)利用氟化修飾方法制備高效、低毒的siRNA載體。我們分別在第五代聚酰胺-胺樹形高分子表面修飾了不同種類的含氟化合物,合成了71個含氟功能基團(tuán)修飾的樹形高分子基因載體。我們篩選了這些氟化修飾載體的siRNA轉(zhuǎn)染效率,其中10個含氟功能基團(tuán)修飾的樹形高分子可實(shí)現(xiàn)高效、低毒的基因沉默效率。有3個含氟基團(tuán)修飾的樹形高分子可在極低氮磷比(氮磷比為0.84：1、0.75：1和1.5：1)條件下達(dá)到80%以上的基因沉默效率,而未修飾的聚酰胺-胺樹形高分子在最佳轉(zhuǎn)染氮磷比為18：1的條件下只能實(shí)現(xiàn)10%以下的基因沉默效率。因此,通過氟化修飾的方法,我們得到了一系列基于樹形高分子的基因載體,可以在低氮磷比條件下實(shí)現(xiàn)高效、低毒的siRNA轉(zhuǎn)染�？偠灾�,本論文基于樹形高分子作為基因轉(zhuǎn)染載體的應(yīng)用過程中存在的轉(zhuǎn)染效率與細(xì)胞毒性的惡性關(guān)聯(lián),通過可逆化合交聯(lián)法、氟化修飾等策略,制備了一系列高效、低毒的樹形高分子基因(DNA和siRNA)載體。
[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.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q78

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