智能响应性多功能纳米基因及药物载体在肿瘤治疗中的应用

发布时间：2018-06-21 13:26

本文选题：基因治疗 + 双硫键　；参考：《华中科技大学》2016年博士论文

【摘要】：第一部分：使用超分子化学技术构建模块化自组装多功能阳离子基因传递系统目的：构建一种基于超分子化学的主-客体自组装多功能大分子阳离子聚合物基因载体系统,评价其性能及生物安全性。方法：首先通过对PCD进行修饰,合成了含有双硫键的大分子ATRP引发剂PCD-SS-BIB;之后通过与DMAEMA的ATRP反应,得到以聚环糊精为主链、含有双硫键的PDMAEMA为侧链的高分子梳状阳离子聚合物PCD-SS-PDMAEMA。通过与金刚烷和叶酸修饰的聚乙二醇(Ad-PEG-FA)自组装形成超分子聚合物PSD-1/PEG-FA。我们对聚合物／质粒DNA的粒径、电势进行了表征以探究聚合物对质粒DNA的压缩绑定能力；通过在HeLa及COS-7细胞中的细胞毒性及溶血实验检测了聚合物的生物相容性；通过荧光素酶报告基因转染评价了聚合物对质粒DNA的转染效果；通过激光共聚焦成像及流式细胞术研究了聚合物／质粒DNA复合物的细胞内吞；通过EGFP的siRNA评价了聚合物对siRNA的转染效果；通过叶酸受体阳性的KB细胞验证了叶酸受体的靶向功能。结果：合成的聚合物可有效地绑定压缩DNA以及干扰RNA(siRNA)形成纳米复合物。通过针对该基因载体系统的理化性能表征及相关生物学实验可以证明,该基因载体具有很好的基因绑定压缩能力、还原敏感性降解能力、低细胞毒性以及特异性的细胞吞噬和优秀的转染效率。结论：基于超分子化学的基因载体合成方法可能为基因载体的“个性化”生物应用提供广泛的前景。第二部分：CD44靶向的智能响应型金-碳纳米球治疗耐药性肿瘤目的：构建基于空心碳纳米球的药物载体,实现对耐药性肿瘤光热-光动力学-化学联合治疗。方法：首先通过聚多巴胺(PDA)包裹在二氧化硅球的表面形成SiO2@PDA复合物。之后,通过PDA对氯金酸钾的还原,得到SiO2@PDA@Au复合物。之后,在高温下碳化PDA并使用氢氟酸去除Si02模板,最终得到了分散有金纳米颗粒的金-碳纳米球(AuC纳米球)。之后通过搭载阿霉素(DOX)及修饰透明质酸,得到了联合治疗纳米药物DOX@AuC@HA。使用红外成像仪对金-碳纳米球在808nnm近红外照射下的光-热转化进行了观察；通过体外释药观察了DOX@AuC@HA对DOX的释放;通过血液相容性及细胞毒性验证了AuC纳米球的生物相容性；通过细胞实验探究了其体外联合治疗效果及逆转耐药性；通过对MCF-7/ADR荷瘤小鼠的联合治疗评价了其体内抗肿瘤效果及体内生物相容性。结果：DOX@AuC@HA具有优秀的光热转化能力、多重敏感的药物释放能力以及CD44靶向的细胞内吞。在近红外光的照射下,DOX@AuC@HA可以通过光热和光动力学的效果极大的促进其所包载的DOX的疗效。在体内实验中,我们也证实了DOX@AuC@HA可以通过系统给药有效的分布到肿瘤部位,且显示出了非常理想的抗肿瘤能力。结论：多功能、多治疗策略联用的DOX@AuC@HA纳米药物可能在治疗耐药性肿瘤上发挥巨大的作用。
[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.
【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R730.5

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