本文選題：基因治療 + 雙硫鍵　； 參考：《華中科技大學》2016年博士論文

【摘要】：第一部分：使用超分子化學技術構建模塊化自組裝多功能陽離子基因傳遞系統(tǒng)目的：構建一種基于超分子化學的主-客體自組裝多功能大分子陽離子聚合物基因載體系統(tǒng),評價其性能及生物安全性。方法：首先通過對PCD進行修飾,合成了含有雙硫鍵的大分子ATRP引發(fā)劑PCD-SS-BIB;之后通過與DMAEMA的ATRP反應,得到以聚環(huán)糊精為主鏈、含有雙硫鍵的PDMAEMA為側鏈的高分子梳狀陽離子聚合物PCD-SS-PDMAEMA。通過與金剛烷和葉酸修飾的聚乙二醇(Ad-PEG-FA)自組裝形成超分子聚合物PSD-1/PEG-FA。我們對聚合物／質粒DNA的粒徑、電勢進行了表征以探究聚合物對質粒DNA的壓縮綁定能力；通過在HeLa及COS-7細胞中的細胞毒性及溶血實驗檢測了聚合物的生物相容性；通過熒光素酶報告基因轉染評價了聚合物對質粒DNA的轉染效果；通過激光共聚焦成像及流式細胞術研究了聚合物／質粒DNA復合物的細胞內吞；通過EGFP的siRNA評價了聚合物對siRNA的轉染效果；通過葉酸受體陽性的KB細胞驗證了葉酸受體的靶向功能。結果：合成的聚合物可有效地綁定壓縮DNA以及干擾RNA(siRNA)形成納米復合物。通過針對該基因載體系統(tǒng)的理化性能表征及相關生物學實驗可以證明,該基因載體具有很好的基因綁定壓縮能力、還原敏感性降解能力、低細胞毒性以及特異性的細胞吞噬和優(yōu)秀的轉染效率。結論：基于超分子化學的基因載體合成方法可能為基因載體的“個性化”生物應用提供廣泛的前景。第二部分：CD44靶向的智能響應型金-碳納米球治療耐藥性腫瘤目的：構建基于空心碳納米球的藥物載體,實現(xiàn)對耐藥性腫瘤光熱-光動力學-化學聯(lián)合治療。方法：首先通過聚多巴胺(PDA)包裹在二氧化硅球的表面形成SiO2@PDA復合物。之后,通過PDA對氯金酸鉀的還原,得到SiO2@PDA@Au復合物。之后,在高溫下碳化PDA并使用氫氟酸去除Si02模板,最終得到了分散有金納米顆粒的金-碳納米球(AuC納米球)。之后通過搭載阿霉素(DOX)及修飾透明質酸,得到了聯(lián)合治療納米藥物DOX@AuC@HA。使用紅外成像儀對金-碳納米球在808nnm近紅外照射下的光-熱轉化進行了觀察；通過體外釋藥觀察了DOX@AuC@HA對DOX的釋放;通過血液相容性及細胞毒性驗證了AuC納米球的生物相容性；通過細胞實驗探究了其體外聯(lián)合治療效果及逆轉耐藥性；通過對MCF-7/ADR荷瘤小鼠的聯(lián)合治療評價了其體內抗腫瘤效果及體內生物相容性。結果：DOX@AuC@HA具有優(yōu)秀的光熱轉化能力、多重敏感的藥物釋放能力以及CD44靶向的細胞內吞。在近紅外光的照射下,DOX@AuC@HA可以通過光熱和光動力學的效果極大的促進其所包載的DOX的療效。在體內實驗中,我們也證實了DOX@AuC@HA可以通過系統(tǒng)給藥有效的分布到腫瘤部位,且顯示出了非常理想的抗腫瘤能力。結論：多功能、多治療策略聯(lián)用的DOX@AuC@HA納米藥物可能在治療耐藥性腫瘤上發(fā)揮巨大的作用。
[Abstract]:Part I: construction of modular multifunctional cationic gene transfer system based on supramolecular chemistry objective: to construct a host-guest self-assembly multi-functional macromolecular cationic polymer gene vector system based on supramolecular chemistry Its performance and biological safety were evaluated. Methods: the macromolecular ATRP initiator PCD-SS-BIBB containing disulfide bond was synthesized by modifying PCD, and then the comb-like cationic polymer PCD-SS-PDMAEMAA was obtained by reacting with the ATRP of DMAEMA and PDMAEMA with PDMAEMA as side chain. A supramolecular polymer PSD-1 / PEG-FAA was synthesized by self-assembly of polyethylene glycol Ad-PEG-FAA modified with adamantane and folic acid. The particle size and potential of polymer / plasmid DNA were characterized to investigate the binding ability of polymer to plasmid DNA, and the biocompatibility of polymer was tested by cytotoxicity and hemolysis test in HeLa and COS-7 cells. The transfection effect of polymer on plasmid DNA was evaluated by luciferase reporter gene transfection, and the endocytosis of polymer / plasmid DNA complex was studied by confocal laser imaging and flow cytometry. The transfection effect of the polymer on siRNA was evaluated by EGFP siRNA, and the targeted function of folate receptor was verified by folate receptor positive KB cells. Results: the synthesized polymers can effectively bind to compress DNA and interfere with RNA-siRNAs to form nanocomposites. According to the physical and chemical properties of the gene vector system and related biological experiments, it can be proved that the gene vector has a good ability of gene binding compression and reduction sensitivity degradation. Low cytotoxicity, specific phagocytosis and excellent transfection efficiency. Conclusion: the method of gene vector synthesis based on supramolecular chemistry may provide a broad prospect for individualized biological application of gene vector. Part II: intelligent responsive gold-carbon nanospheres targeting CD44 for the treatment of drug-resistant tumors objective: to construct a drug carrier based on hollow carbon nanospheres and to achieve photothermal-photodynamic chemotherapeutic combined therapy for drug-resistant tumors. Methods: at first, Sio _ 2 @ PDA complex was formed on the surface of silica sphere by polydopamine PDA-encapsulated. After that, through the reduction of potassium chloraurate by PDA, the Sio _ 2 @ PDA-R au complex was obtained. After carbonation of PDA at high temperature and removal of Si02 template with hydrofluoric acid, au C nanospheres dispersed with gold nanoparticles were obtained. After that, the combination therapy of doxobenzene (DOX) and modified hyaluronic acid (HA) was obtained. The photothermal transformation of gold-carbon nanospheres irradiated by 808nnm near infrared irradiation was observed by infrared imager, the release of DOX from DOXR was observed by drug release in vitro, and the biocompatibility of AUC nanospheres was verified by blood compatibility and cytotoxicity. The in vitro combined treatment and reversal of drug resistance were investigated by cell experiments, and the anti-tumor effect and biocompatibility of MCF-7 / ADR bearing mice were evaluated. Results the results showed that: 1. The ability to convert photoheat, the ability of drug release and the endocytosis of CD44 were excellent. DOX @ AuC@ HA can greatly promote the efficacy of DOX by photothermal and photodynamic effects under near-infrared light irradiation. In vivo experiments, we also confirmed that DOX @ AuC@ HA can be effectively distributed to the tumor site through systemic administration, and has shown a very good anti-tumor ability. Conclusion: the multifunctional, multitherapy strategy combined with DOX @ AuC@ HA nanopharmaceuticals may play an important role in the treatment of drug-resistant tumors.
【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：R730.5

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