【摘要】：陽離子高分子作為一類重要的非病毒基因遞送載體,具有很多優(yōu)于病毒類載體的特點:無免疫原性、無致癌性、易于合成且穩(wěn)定,因此在過去的幾十年里得到巨大的發(fā)展。然而,陽離子高分子在實際應(yīng)用過程中存在著轉(zhuǎn)染效率和細胞毒性之間的惡性關(guān)聯(lián):(1)轉(zhuǎn)染效率高的陽離子高分子載體,細胞毒性也高;(2)細胞毒性低的陽離子高分子載體,轉(zhuǎn)染效率也低。因此打破這一惡性關(guān)聯(lián),開發(fā)出高效、低毒的陽離子高分子載體,成為基因治療這一科學領(lǐng)域亟待解決的重要問題。為此我們提出一個解決思路:構(gòu)建基于陽離子樹形高分子的超分子體系,從而獲得高效、低毒的高分子復合物。本文提出以下兩種方法來構(gòu)建超分子體系:(1)開發(fā)一種核-殼結(jié)構(gòu)的高分子復合物:用高代數(shù)的樹形高分子作為核心高分子,在低氮磷比下壓縮質(zhì)粒DNA以抵消其正電性,然后以低分子量的陽離子聚合物作為外殼高分子,包裹在外部。我們選用三種高代數(shù)(Generation,G)的聚酰胺-胺(PAMAM)樹形高分子作為核心高分子,然后分別選用低分子量的線性聚乙烯亞胺(LPEI)、支狀聚乙烯亞胺(BPEI)和G2PAMAM作為外殼做測試。結(jié)果發(fā)現(xiàn)復合物以G8PAMAM為核心高分子、LPEI為外殼高分子時,表現(xiàn)出較高的基因轉(zhuǎn)染效率,并且細胞毒性很低。進一步的轉(zhuǎn)染機制研究結(jié)果表明,這種核-殼結(jié)構(gòu)有利于復合物在細胞質(zhì)中釋放DNA。(2)利用化學交聯(lián)策略:將低代數(shù)的G2PAMAM與富勒烯C60化學交聯(lián),得到結(jié)構(gòu)均勻的納米顆粒,基因轉(zhuǎn)染效率大為提高,且細胞安全性較好。進一步的轉(zhuǎn)染機制研究表明,該交聯(lián)產(chǎn)物大大提高了細胞對基因的攝入能力。本論文基于陽離子樹形高分子提出了兩種獨特的策略,獲得兩種易于合成的轉(zhuǎn)染復合物G8/LPEI/DNA和C60-G2/DNA,具有較高的細胞轉(zhuǎn)染效率和較低的細胞毒性。后續(xù)我們將進一步評估這兩種轉(zhuǎn)染復合物的體內(nèi)遞送效率,并探索G2 PAMAM與富勒烯C60交聯(lián)產(chǎn)物的光動力學治療作用,為基因治療聯(lián)合光動力學治療奠定良好的材料學基礎(chǔ)。
[Abstract]:As a kind of important non-viral gene delivery vector, cationic polymer has many advantages over virus-like vectors: no immunogenicity, no carcinogenicity, easy synthesis and stability, so it has been greatly developed in the past few decades. However, there is a malignant correlation between transfection efficiency and cytotoxicity of cationic polymer in practical application: (1) cationic polymer carrier with high transfection efficiency and high cytotoxicity; (2) cationic polymer carrier with low cytotoxicity; The transfection efficiency was also low. Therefore, breaking this malignant association and developing cationic polymer carriers with high efficiency and low toxicity has become an important problem to be solved in the field of gene therapy. Therefore, we propose a solution: to construct a supramolecular system based on cationic dendrimer, so as to obtain highly efficient and low toxic polymer complexes. In this paper, the following two methods are proposed to construct supramolecular system: (1) to develop a core-shell polymer complex: high algebraic dendritic polymer is used as the core polymer, and the plasmid DNA is compressed at low nitrogen / phosphorus ratio to counteract its positive electrical properties. Then the cationic polymer with low molecular weight was used as the outer polymer. Three kinds of high algebra (Generation,G) polyamide-amine (PAMAM) dendrimer were selected as core polymers, and the low molecular weight linear polyethylene imine (LPEI), branched polyimide (BPEI) and G2PAMAM were used as the shell respectively. The results showed that when G8PAMAM was used as the core polymer and LPEI was used as shell polymer, the efficiency of gene transfection was higher and the cytotoxicity was very low. Further studies on the transfection mechanism showed that the core-shell structure was conducive to the release of DNA. (2) from the cytoplasm of the complex. (2) the low algebraic G2PAMAM was chemically crosslinked with fullerene C60 to obtain homogeneous nanoparticles. The efficiency of gene transfection was greatly improved, and the cell safety was better. Further studies on the transfection mechanism showed that the cross-linked product greatly increased the ability of the cells to ingest genes. Based on cationic dendrimer, two novel transfection complexes, G8/LPEI/DNA and C60-G _ 2 / DNA, have been proposed in this paper, which have high transfection efficiency and low cytotoxicity. We will further evaluate the delivery efficiency of these two transfection complexes in vivo, and explore the photodynamic effects of G2 PAMAM and fullerene C60 crosslinking products, which will lay a good material foundation for gene therapy combined with photodynamic therapy.
【學位授予單位】：華東師范大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R450

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本文編號：2201419