新型聚阳离子基因载体模型的构建
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摘要
本论文的研究内容包括两个部分:第一部分探索适用于氧阴离子聚合的不同结构的引发剂与单体,探讨了氧阴离子聚合的聚合机理;第二部分利用氧阴离子聚合与层层组装技术的优势,设计一种新型聚阳离子基因载体模型,并对其进行系统的研究。
(1)利用氧阴离子聚合原理,分别采用胆固醇(Cholesterol)和单甲基封端的聚乙二醇(MePEG)与KH反应作为引发剂体系,单体为甲基丙烯酸-2-(N,N-甲氨基)乙酯(DMAEMA)和甲基丙烯酸叔丁酯(tBMA),成功合成结构明确的功能性聚合物Chol-PDMAEMA和MePEO-b-PtBMA。通过~1H NMR和GPC测试分别获得聚合物的分子量及分子量分布数据。结果表明,所得聚合物的分子量分布较窄,聚合物分子量的实验值与理论值较吻合。本研究首次发现甲基丙烯酸叔丁酯同样适用于氧阴离子聚合反应,修正了以前文献中对于氧阴离子聚合机理的认识,为开发适合氧阴离子聚合的单体提供更有力的支撑。
(2)设计构建一种新型聚阳离子型因载体模型。该模型以Chol-PDMAEMA为聚阳离子,以功能化的MePEG2000-b-PMAA_(SH)为聚阴离子,构建基因载体模型,其步骤分为以下三步:(i)将DNA与聚阳离子Chol-PDMAEMA_(30)复合,形成表面带正电荷的Chol-PDMAEMA_(30)/DNA复合物;(ii)为了克服复合物表面带正电荷所引起的非特异性清除,使用功能化的聚阴离子MePEG2000-b-PMAA_(SH),通过静电力作用包裹Chol-PDMAEMA_(30)/DNA复合物,屏蔽表面的正电荷,获得MePEG2000-b-PMAA_(SH)/Chol-PDMAEMA_(30)/DNA复合物;(iii)为提高载体的稳定性,向上述复合物溶液中加入过氧化氢,氧化MePEG2000-b-PMAA_(SH)中的巯基(-SH),使巯基之间形成二硫键(S-S),.固定壳层。通过对每一步复合过程的Zeta电位、粒径与透射电镜的研究,表明所设计的载体模型符合预期的结果。通过临界胶束浓度测试、凝胶电泳实验和溴已锭取代实验,研究低分子量聚阳离子Chol-PDMAEMA_(30)与DNA结合能力以及Chol-PDMAEMA_(30)/DNA复合物的稳定性,研究结果表明,Chol-PDMAEMA_(30)是一种高效的聚阳离子型基因载体。通过凝胶电泳实验与溴已锭取代实验,研究在使用该模型构建基因载体中一个至关重要的协同链交换问题,定性及定量化的研究结果表明当MePEG2000-b-PMAA_(SH)中[MAA]与Chol-PDMAEMA中[DMAEMA]的摩尔量比为1:1时较为合适。通过体外模拟,研究载体在细胞外与细胞内环境下的理化性能。研究结果表明,这类复合基因载体在血液传输过程中是稳定的,但是,当进入到细胞内部,由于细胞内的还原作用,二硫键断裂,载体的胶囊结构被破坏,DNA被释放。
In the present study, for one thing, a new type of well-defined WSLP gene carrier consisting of hydrophilic poly[(dimethylamino)ethyl methacrylate] (PDMAEMA) and hydrophobic cholesterol (Chol-PDMAEMA_(30)) was prepared by a one-step synthesis route via oxyanion-initiated polymerization; for another, a facile route for DNA encapsulation in triggered intracellular degradable polymer microcapsules has been achieved via electrostatic interaction, using a polycation, Chol-PDMAEMA_(30), along with a polyanion named MePEG2000-block-poly- (methacrylic acid) carring partial thiol groups (MePEG2000-b-PMAA_(SH)), which was also prepared via oxyanion-initiated polymerization. The encapsulation procedure involves three steps: (i) DNA was first complexed with the polycation (Chol-PDMAEMA_(30)); (ii) To overcome the limitations of the positively charged complexes, MePEG2000-b-PMAA_(SH), was used to coat the Chol-PDMAEMA_(30)/DNA complexes by electrostatic interaction; and (iii) the complexes were stabilized by oxidizing the thiol groups to form bridging disulfide linkages between the MePEG2000-b-PMAA_(SH) chains. The properties of Chol-PDMAEMA_(30), and the interactions between every pair among calf thymus DNA, Chol-PDMAEMA_(30), and MePEG2000-b-PMAA_(SH) were studied by critical micelle concentration determination, agarose gel retardation assay and ethidium bromide displacement assay. The results indicate that the prepared microcapsules may remain stable during systemic circulation, but degrade and release the carried DNA in a cellular reducing environment. The studies on chain-exchanging reaction indicate that the excess MePEG2000-b-PMAA_(SH) will lead to the aborted binding of Chol-PDMAEMA_(30) with DNA and the amount of MePEG2000-b-PMAA_(SH) is appropriate when the [MAA]/[DMAEMA] molar ratio is in the range of 1.0-1.5. However, the results of further investigation showed that the exchange reaction may be beneficial for the DNA release when the disulfide linkages are destroyed in cells. Furthermore, the biophysical properties of the microcapsule have been investigated byζ-potential, laser light scattering, and transmission electron microscopy (TEM) measurements.
(1)利用氧阴离子聚合原理,分别采用胆固醇(Cholesterol)和单甲基封端的聚乙二醇(MePEG)与KH反应作为引发剂体系,单体为甲基丙烯酸-2-(N,N-甲氨基)乙酯(DMAEMA)和甲基丙烯酸叔丁酯(tBMA),成功合成结构明确的功能性聚合物Chol-PDMAEMA和MePEO-b-PtBMA。通过~1H NMR和GPC测试分别获得聚合物的分子量及分子量分布数据。结果表明,所得聚合物的分子量分布较窄,聚合物分子量的实验值与理论值较吻合。本研究首次发现甲基丙烯酸叔丁酯同样适用于氧阴离子聚合反应,修正了以前文献中对于氧阴离子聚合机理的认识,为开发适合氧阴离子聚合的单体提供更有力的支撑。
(2)设计构建一种新型聚阳离子型因载体模型。该模型以Chol-PDMAEMA为聚阳离子,以功能化的MePEG2000-b-PMAA_(SH)为聚阴离子,构建基因载体模型,其步骤分为以下三步:(i)将DNA与聚阳离子Chol-PDMAEMA_(30)复合,形成表面带正电荷的Chol-PDMAEMA_(30)/DNA复合物;(ii)为了克服复合物表面带正电荷所引起的非特异性清除,使用功能化的聚阴离子MePEG2000-b-PMAA_(SH),通过静电力作用包裹Chol-PDMAEMA_(30)/DNA复合物,屏蔽表面的正电荷,获得MePEG2000-b-PMAA_(SH)/Chol-PDMAEMA_(30)/DNA复合物;(iii)为提高载体的稳定性,向上述复合物溶液中加入过氧化氢,氧化MePEG2000-b-PMAA_(SH)中的巯基(-SH),使巯基之间形成二硫键(S-S),.固定壳层。通过对每一步复合过程的Zeta电位、粒径与透射电镜的研究,表明所设计的载体模型符合预期的结果。通过临界胶束浓度测试、凝胶电泳实验和溴已锭取代实验,研究低分子量聚阳离子Chol-PDMAEMA_(30)与DNA结合能力以及Chol-PDMAEMA_(30)/DNA复合物的稳定性,研究结果表明,Chol-PDMAEMA_(30)是一种高效的聚阳离子型基因载体。通过凝胶电泳实验与溴已锭取代实验,研究在使用该模型构建基因载体中一个至关重要的协同链交换问题,定性及定量化的研究结果表明当MePEG2000-b-PMAA_(SH)中[MAA]与Chol-PDMAEMA中[DMAEMA]的摩尔量比为1:1时较为合适。通过体外模拟,研究载体在细胞外与细胞内环境下的理化性能。研究结果表明,这类复合基因载体在血液传输过程中是稳定的,但是,当进入到细胞内部,由于细胞内的还原作用,二硫键断裂,载体的胶囊结构被破坏,DNA被释放。
In the present study, for one thing, a new type of well-defined WSLP gene carrier consisting of hydrophilic poly[(dimethylamino)ethyl methacrylate] (PDMAEMA) and hydrophobic cholesterol (Chol-PDMAEMA_(30)) was prepared by a one-step synthesis route via oxyanion-initiated polymerization; for another, a facile route for DNA encapsulation in triggered intracellular degradable polymer microcapsules has been achieved via electrostatic interaction, using a polycation, Chol-PDMAEMA_(30), along with a polyanion named MePEG2000-block-poly- (methacrylic acid) carring partial thiol groups (MePEG2000-b-PMAA_(SH)), which was also prepared via oxyanion-initiated polymerization. The encapsulation procedure involves three steps: (i) DNA was first complexed with the polycation (Chol-PDMAEMA_(30)); (ii) To overcome the limitations of the positively charged complexes, MePEG2000-b-PMAA_(SH), was used to coat the Chol-PDMAEMA_(30)/DNA complexes by electrostatic interaction; and (iii) the complexes were stabilized by oxidizing the thiol groups to form bridging disulfide linkages between the MePEG2000-b-PMAA_(SH) chains. The properties of Chol-PDMAEMA_(30), and the interactions between every pair among calf thymus DNA, Chol-PDMAEMA_(30), and MePEG2000-b-PMAA_(SH) were studied by critical micelle concentration determination, agarose gel retardation assay and ethidium bromide displacement assay. The results indicate that the prepared microcapsules may remain stable during systemic circulation, but degrade and release the carried DNA in a cellular reducing environment. The studies on chain-exchanging reaction indicate that the excess MePEG2000-b-PMAA_(SH) will lead to the aborted binding of Chol-PDMAEMA_(30) with DNA and the amount of MePEG2000-b-PMAA_(SH) is appropriate when the [MAA]/[DMAEMA] molar ratio is in the range of 1.0-1.5. However, the results of further investigation showed that the exchange reaction may be beneficial for the DNA release when the disulfide linkages are destroyed in cells. Furthermore, the biophysical properties of the microcapsule have been investigated byζ-potential, laser light scattering, and transmission electron microscopy (TEM) measurements.
引文
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