【摘要】：聚乙烯亞胺（PEI）作為最典型的陽離子聚合物之一，目前已經(jīng)廣泛用作基因轉染的載體，其中尤以分子量為25KDa的超支化PEI（b-PEI），以其優(yōu)異的轉染性能被稱為基因遞送的“黃金標準”。但由于b-PEI25K較高的陽性電荷密度，導致其細胞毒性大，血液相容性差等缺點，從而嚴重制約了其進一步的應用。天然化合物如氨基酸、蛋白質及糖胺聚糖，往往具有良好的生物相容性、較低的免疫源性及細胞毒性，部分天然化合物（如硫酸軟骨素）還能夠特異性結合腫瘤細胞表面的抗原分子（CD-44），，是PEI修飾改性的良好配體選擇并能夠實現(xiàn)靶向性遞送。本論文結合天然化合物的多種優(yōu)勢，設計并合成了三種以天然化合物改性的b-PEI25K衍生物，并將三種衍生物分別與DNAzyme、pDNA、microRNA等不同基因藥物組裝，重點評價了衍生物的理化性質、衍生物與基因藥物的組裝及相互作用、載體的生物相容性、細胞毒性及載體在體外水平的轉染性能，進而考察了基因藥物在體外轉染后對細胞的影響及藥效。本文的研究重點分為以下幾個方面: （1）采用EDC/NHS催化的方法，以N-乙酰-亮氨酸為原料，與b-PEI25K的伯氨基進行偶聯(lián)，合成了不同比例的疏水性修飾的PEI衍生物，基因載體N-Ac-L-Leu-PEI。對合成載體的結構及理化性質進行了表征，檢測了衍生物載體的細胞毒性和生物相容性，及其與pDNA、DNAzyme的結合能力，評價了衍生物載體與DNAzyme的復合物的胞內行為及對細胞增殖、遷移及浸潤的影響。相關實驗結果均表明衍生物具備較低的細胞毒性及良好的轉染效率和生物相容性，轉染DNAzyme進入細胞后有效的抑制了腫瘤細胞的增殖和遷移。 （2）通過化學鍵合的手段，以具有紅色熒光的京尼平作為交聯(lián)劑，將低毒性的嗜熱組蛋白與高轉染效率的PEI25K進行鍵合，合成了低細胞毒性，高轉染效率的具有協(xié)同效應的蛋白質-高分子雜合基因傳輸載體。對組蛋白－PEI雜合基因載體進行了結構與性質的表征，驗證了蛋白質與高分子的有效鍵合，并檢測了新型載體與pDNA的組裝能力、細胞毒性及轉染效率，實驗結果表明該新型雜合基因載體具備良好的DNA包覆能力，基因轉染效率高以及較低的細胞毒性。 （3）以天然糖胺聚糖硫酸軟骨素為配體通過Michael加成反應與b-PEI25K實現(xiàn)偶聯(lián)，利用硫酸軟骨素能夠特異性識別腫瘤基質中高表達的CD44受體的特點，構建了低毒、高效、具有腫瘤靶向能力的新型基因遞送體系，并實現(xiàn)抑癌microRNA藥物miR-34a對腫瘤細胞的靶向遞送。對該載體的理化性質、載藥能力、細胞毒性及靶向能力進行了評估，并對轉染基因藥物進入細胞后的胞內定位、相關蛋白表達量及細胞遷移能力進行了測定。實驗結果表明該新型靶向型基因載體通過CD44介導的細胞內吞能夠有效的將miR-34a遞送到腫瘤細胞內，通過誘導腫瘤細胞的凋亡及抑制腫瘤細胞的遷移達到雙重抗瘤的效果。 綜上所述，本論文中合成的基于PEI與天然化合物的多種基因載體體系，在具有良好的生物相容性、較低細胞毒性的同時能夠保持高的轉染效率，并能有效介導所搭載的基因藥物在胞內發(fā)揮生物學功能，因此可望成為高效安全的新型基因載體。
[Abstract]:Polyethylenimide (PEI), as one of the most typical cationic polymers, has been widely used as a vector for gene transfection, especially hyperbranched PEI (b-PEI) with molecular weight of 25 KDa, which is called the "golden standard" for gene delivery because of its excellent transfection performance. Natural compounds, such as amino acids, proteins and glycosaminoglycans, often have good biocompatibility, low immunogenicity and cytotoxicity, and some natural compounds (such as chondroitin sulfate) can specifically bind to antigens on the surface of tumor cells. CD-44 is a good ligand selection for PEI modification and can achieve targeted delivery. In this paper, three kinds of b-PEI 25K derivatives modified by natural compounds were designed and synthesized, and three derivatives were assembled with DNA zyme, pDNA, microRNA and other gene drugs, with emphasis on the evaluation of derivatives. The physical and chemical properties, the assembly and interaction of derivatives and gene drugs, the biocompatibility of the carriers, cytotoxicity and the transfection performance of the carriers in vitro were investigated. The effects of gene drugs on cells after transfection in vitro were investigated.
(1) Hydrophobically modified PEI derivatives with different proportions were synthesized by coupling N-acetyl-leucine with primary amino group of b-PEI 25K using EDC/NHS as catalyst. The structure and physicochemical properties of the synthesized carriers were characterized, and the cytotoxicity and biocompatibility of the carriers were tested. The binding ability of the derivatives with pDNA and DNA zyme was evaluated. The intracellular behavior of the derivatives with DNA zyme and their effects on cell proliferation, migration and infiltration were evaluated. Cell proliferation and migration.
(2) Protein-macromolecule hybrid gene delivery vectors with low cytotoxicity and high transfection efficiency were synthesized by chemical bonding and crosslinking with genipin with red fluorescence as crosslinking agent. Histone-PEI hybrid gene delivery vectors were introduced. The structure and properties of the hybrid gene vector were characterized to verify the effective binding between protein and polymer. The assembly ability, cytotoxicity and transfection efficiency of the new vector with pDNA were tested. The results showed that the hybrid gene vector had good DNA coating ability, high gene transfection efficiency and low cytotoxicity.
(3) Using natural glycosaminoglycan chondroitin sulfate as ligand to couple with b-PEI25K by Michael addition reaction, a novel gene delivery system with low toxicity, high efficiency and tumor targeting ability was constructed by using chondroitin sulfate as a specific recognition of CD44 receptor in tumor matrix, and the anti-cancer microRNA drug microRNA microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs microRNAs micro The physical and chemical properties, drug loading capacity, cytotoxicity and targeting ability of the vector were evaluated. The localization, expression of related proteins and cell migration ability of the transfected gene drug after entering the cell were determined. The experimental results showed that the novel targeting gene vector was fine mediated by CD44. Intracellular endocytosis can effectively deliver microRNA-34a to tumor cells and achieve dual antitumor effects by inducing apoptosis and inhibiting migration of tumor cells.
In summary, the PEI-based gene delivery systems synthesized in this paper have good biocompatibility, low cytotoxicity and high transfection efficiency, and can effectively mediate the biological functions of the gene drugs carried in the cells. Therefore, PEI-based gene delivery systems are expected to be a new type of genes with high efficiency and safety. Carrier.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R91;R914.5

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