【作者】 唐凌燕；

【导师】 王均；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2009， 硕士

【摘要】 研究和开发具有良好性能的药物和基因输送载体是目前生物医学领域的重要研究方向。本论文针对药物和基因的输送设计合成了两种性能优越的载体材料,并对它们的性能进行了研究。我们设计合成了一种还原响应性的纳米药物载体,载体材料是由二硫键连接的两亲性嵌段聚合物PCL-SS-PEEP,含有疏水性聚己内酯嵌段和亲水性聚2-乙氧基-2-氧-1,3,2-二氧磷杂环戊烷嵌段。载体材料的化学结构经凝胶渗透色谱(GPC)和1H NMR等表征手段验证。PCL-SS-PEEP在水溶液中自组装形成胶束纳米粒,芘荧光探针法测得其临界胶束浓度为3.1×10-3 mg mL-1,透射电子显微镜(TEM)观察和动态光散射(DLS)检测表明胶束的粒径在100 nm左右且分布均匀。该胶束与10 mM谷胱甘肽(GSH)共培养时,DLS检测颗粒粒径随时间延长显著增大,显示在GSH作用下载体聚合物二硫键断裂破坏了胶束的结构,并因此造成团聚。通过疏水相互作用可以将阿霉素(DOX)包载到胶束内核中,而且DOX从胶束的释放具有GSH浓度响应性,在较高GSH浓度下DOX的释放更快。在细胞实验中,用谷胱甘肽单乙酯(GSH-OEt)处理A549细胞,构建了胞内富集GSH的细胞模型,将载有DOX的胶束纳米粒和A549细胞培养,激光共聚焦和流式细胞检测表明,经GSH-OEt处理的A549细胞内阿霉素荧光比未经GSH-OEt处理的A549细胞增强,表明GSH-OEt处理的A549细胞内高的GSH浓度造成更多的DOX在胞内快速释放。此外,在相同DOX剂量下,和载有DOX的胶束纳米粒共培养的细胞存活率随加入GSH-OEt浓度的增加而下降,也说明PCL-SS-PEEP胶束的GSH响应性的药物释放行为。这种智能化的胞内药物输送和释放为药物输送的纳米载体的设计提供了新的思路。在基因输送载体的研究中,通过聚乙烯亚胺和2-乙氧基-2-氧-1,3,2-二氧磷杂环戊烷的化学反应获得了具有不同磷酸酯修饰度的聚乙烯亚胺衍生物(PEI-EEP),利用1H和31P NMR表征了聚合物的结构并初步推测了反应进行的机理。这一系列PEI-EEP聚合物表现出与PEI类似的缓冲能力,在较低的N/P比下完全结合DNA。我们用DLS的手段研究了不同修饰度的PEI-EEP与DNA在不同N/P下结合形成复合物的粒径和电势的变化趋势,同时用噻唑蓝(MTT)细胞活性证明PEI-EEP聚合物与PEI相比,对HEK293和HeLa细胞均显示低的细胞毒性,而且随修饰度的增加而下降。PEI-PEEP对HEK293和HeLa的体外转染实验结果表明其转染效率与修饰度密切相关,每个PEI分子中含EEP修饰度为52和75的PEI-EEP聚合物表现出比PEI高的转染效率,当EEP修饰度增加至116个时,聚合物的转染效率则显著下降。此外,与无血清时相比,PEI-EEP聚合物在血清存在时表现出更高的转染效率。综合上述,PEI-EEP聚合物可以同时表现出高的转染效率和低的细胞毒性,表明PEI-EEP有潜力成为基因输送的载体材料。更多还原

【Abstract】 Development of drug and gene delivery carriers with fine properties is a hot topic of biomedicine research. In the thesis, two polymers with unique properties were designed and synthesized as the carriers of drug and gene delivery systems, respectively. The structures and properties of the two polymers were well characterized.A novel glutathione-responsive amphiphilic block copolymer consisting of polycaprolactone and polyphosphoester with intervening disulfide bond, termed as PCL-SS-PEEP, was synthesized. The block copolymer was well characterized by 1H NMR and gel permeation chromatograph. The amphiphilic copolymer formed micelles in aqueous solution and the critical micelle concentration was 3.1×10-3 mg mL-1. Transmition electron microscopy and dynamic light scattering (DLS) measurements demonstrated that the micelles were relatively uniform in size with the average diameter ca. 100 nm. The micelles displayed glutathione-dependent size changes according to DLS measurement. Doxorubicin [1] was encapsulated in the micelles and its in vitro release profile demonstrated that more DOX was released at a faster rate in the presence of glutathione (GSH). At cellular level, the cells were pretreated with glutathione monoester (GSH-OEt) at different concentrations to manipulate the intracellular GSH concentration. The confocal laser scanning microscopic and flow cytometric analyses indicated that more intensive fluorescence of DOX were detected in cells pretreated by GSH-OEt due to the GSH-triggered DOX release. The DOX-loaded micelles also exhibited higher cytotoxicity in GSH-OEt-pretreated A549 cells. This indicated that the GSH-responsive PCL-SS-PEEP micelles are promising for intracellular drug delivery and it also provides a new paradigm for designing drug delivery vehicles.Cyclic phosphate monomer ethyl ethylene phosphate (EEP) modified poly(ethylenimine) (PEI), denoted as PEI-EEP, was synthesized for gene delivery. Three PEI-EEP polymers were synthesized and their structures were characterized by 1H and 31P NMR methods. The buffering capabilities of PEI-EEPs were examined by acid–base titration and the titration profiles showed that all of the PEI-EEPs had the similar buffering capability as unmodified PEI. Physiochemical characteristics of PEI-EEP/DNA complexes were analyzed by agarose gel electrophoresis, and particle size and zeta potential measurements. All of the PEI-EEPs were able to condense DNA efficiently at N/P ratios higher than 0.5:1. The particle sizes of PEI-EEPs/DNA complexes were around 160–250 nm, while the surface charges were around 30-50 mV at the N/P ratios ranging from 10:1 to 50:1. In vitro cell viability and transfection ability were evaluated in HEK293 and HeLa cells using PEI as the control. The cytotoxicity of PEI-EEPs and PEI-EEPs/DNA complexes were lower than that of PEI and its complexes with DNA. The in vitro transfection experiments indicated that the transfection efficiency of PEI-EEP/DNA complexes were related to the modification degrees by the phosphate. When the phosphate units per PEI were 52 and 75, the PEI-EEP/DNA complexes exhibited comparable or even higher transfection ability than PEI/DNA complex did at its optimal N/P ratio of 10:1 in the absence of serum. However, when the phosphate units increased to 116 per PEI, the transfection efficiency dramatically reduced.更多还原