纳米壳聚糖衍生物金属配合物的制备及其与DNA作用的研究
摘要
本论文由四部分组成：
第一章 文献综述，甲壳素／壳聚糖及其衍生物在药物研究中的应用
甲壳素／壳聚糖及其衍生物由于良好的物理化学性质和医药功能，在药物研究领域中得到了广泛的应用。文中主要从甲壳素／壳聚糖及其衍生物的医药功能、药理活性、作为药物载体和在基因治疗中作为基因载体四个方面集中进行了综述。
第二章 纳米壳聚糖金属配合物的制备及其与DNA作用的研究
采用氧化降解法将壳聚糖降解制得两种不同分子量的水溶性纳米壳聚糖COS1(20000)和COS2(19000)，并将其与Cu(Ⅱ)、Ni(Ⅱ)、Zn(Ⅱ)、Co(Ⅱ)四种金属配位合成了一系列纳米壳聚糖金属配合物。采用凝胶色谱法、元素分析、金属含量测定和红外光谱方法对配合物的结构和组成进行了表征。
采用紫外光谱、荧光光谱和琼脂糖凝胶电泳三种分析手段首次研究了COS—Met系列壳聚糖金属配合物与DNA的作用，并提出了可能的作用机理和模式。认为此类壳聚糖金属配合物与DNA作用时存在静电结合和嵌入两种作用模式：首先壳聚糖骨架上带正电的自由氨基与DNA骨架上带负电的磷酸基团有静电结合作用，使DNA螺旋双链在轴向方向上收缩，构象发生变化；继而配合物的一部分以嵌入模式插入DNA双螺旋内部碱基对之间，从而使DNA的二级结构受到不同程度的损伤。在对壳聚糖配合与DNA作用时配合物结构对其影响的研究中发现，壳聚糖的分子量、溶液中游离氨基的浓度以及壳聚糖骨架上金属的密度对其与DNA的作用模式也有不同程度的影响。
第三章 纳米壳聚糖水杨醛希夫碱金属配合物的合成及其与DNA作用的研究
将水溶性纳米壳聚糖COS3(19000)和)和COS4(4000)经水杨醛改性后与Cu(Ⅱ)、Ni(Ⅱ)、Zn(Ⅱ)、Co(Ⅱ)四种金属配位合成了一系列壳聚糖水杨醛希夫碱金属配合物。并对配合物的结构和组成进行了表征。
采用紫外光谱、荧光光谱和琼脂糖凝胶电泳三种分析手段首次研究了COS
一Met系列壳聚糖金属配合物与DNA的作用，并提出了可能的作用机理和模式。
结果表明此类壳聚糖金属配合物与DNA作用时存在静电结合和嵌入两种作用模
式。由于COS分子量的降低和配合物平面性增强使其与DNA的作用程度明显增
强，同时，考察了纳米壳聚糖的分子量、溶液中游离氨基的浓度以及壳聚糖骨架
上金属的密度对COS一Sal一Met与DNA的作用模式和作用程度的影响。
第四章纳米壳聚糖卜毗咤醛希夫碱金属配合物的合成及其与DNA作用的研究
首次合成了一系列纳米壳聚糖卜毗陡醛希夫碱金属(Cu、Ni、Zn、Co)配
合物，并对配合物的结构和组成进行了表征。
采用紫外光谱，荧光光谱和琼脂糖凝胶电泳三种分析手段首次研究了COS
一P一Mct系列壳聚糖金属配合物与DNA的作用。此类壳聚糖金属配合物与
DNA作用时存在静电结合模式。同时DNA也有不同程度的降解，其机理还有其
他作用模式有待于进一步研究。
Chitin/chitosan and their derivatives have been various applied in the pharmaceutical field, due to theirs good physical chemistry property and pharmaceutical function. In recently, chitin and chitosan of low molecular weight have been significant noted because of their many encouraging results and specific features. In this review, it is focus on the achievement in the field of pharmaceutical application, such as pharmaceutical function and biological activity of chitin/ chitosan and theirs derivatives, the application as drug carrier material and as gene delivery vector in gene therapy.
Chitosan was depolymerized with oxidation method, which afforded nano-chitosan of low molecular weights (COS Mw=20 000, Mw=19 000, 4 000). Nano-chitosan metal complexes (COS-Met), Nano-chitosan salicylaldehyde Schiff base metal complexes (COS-Sal-Met) and a series of novel Nano-chitosanm a-pyridine-carboxaldehyde Schiff base metal complexes (COS-P-Met) with Cu (II), Ni( II ), Zn( II), Co( II) were synthesized. The nano-chitosans and their complexes were characterized by means of gel permeation chromatography (GPC), IR spectra, Elemental analysis and ICP. The interaction of DNA with these nano-chitosan metal complexes was studied by UV/Vis, fluorescent probe technicality and electrophoresis analysis at the first time. The possible interaction mechanism and mode were discussed.
The results indicate that interaction between the nano-chitosan metal complex (COS-Met), Nano-chitosan salicylaldehyde Schiff base metal complexes (COS-Sal-Met) with DNA has two modes. Firstly, the free amine positive charged of nano-chitosan backbone electrostatic bind to negative charged phosphate backbone at the periphery of the double helix DNA, then double helix DNA contracted at axial and its conformation was changed. Secondly, a portion of ligands intercalated between the base pair of the DNA duplex helix cause the secondary structure of the DNA destroyed. Furthermore, the increasing water solubility and the plenary complex structure of
(COS-Sal-Met) improved theirs reaction activity. The nano-chitosan a-pyridine-carboxaldehyde Schiff base metal complexes (COS-P-Met) interacting with DNA has electrostatic binding mode being observed. It should be further studied to determine whether there are other modes existed. At the same time, the influence on their interaction degree and mode of three kinds of nano-chitosan metal complexes chemical structure, such as the molecular weights of nano-chitosan, the content of free amine and metal of nano-chitosan metal complexes, were determined.
第一章 文献综述，甲壳素／壳聚糖及其衍生物在药物研究中的应用
甲壳素／壳聚糖及其衍生物由于良好的物理化学性质和医药功能，在药物研究领域中得到了广泛的应用。文中主要从甲壳素／壳聚糖及其衍生物的医药功能、药理活性、作为药物载体和在基因治疗中作为基因载体四个方面集中进行了综述。
第二章 纳米壳聚糖金属配合物的制备及其与DNA作用的研究
采用氧化降解法将壳聚糖降解制得两种不同分子量的水溶性纳米壳聚糖COS1(20000)和COS2(19000)，并将其与Cu(Ⅱ)、Ni(Ⅱ)、Zn(Ⅱ)、Co(Ⅱ)四种金属配位合成了一系列纳米壳聚糖金属配合物。采用凝胶色谱法、元素分析、金属含量测定和红外光谱方法对配合物的结构和组成进行了表征。
采用紫外光谱、荧光光谱和琼脂糖凝胶电泳三种分析手段首次研究了COS—Met系列壳聚糖金属配合物与DNA的作用，并提出了可能的作用机理和模式。认为此类壳聚糖金属配合物与DNA作用时存在静电结合和嵌入两种作用模式：首先壳聚糖骨架上带正电的自由氨基与DNA骨架上带负电的磷酸基团有静电结合作用，使DNA螺旋双链在轴向方向上收缩，构象发生变化；继而配合物的一部分以嵌入模式插入DNA双螺旋内部碱基对之间，从而使DNA的二级结构受到不同程度的损伤。在对壳聚糖配合与DNA作用时配合物结构对其影响的研究中发现，壳聚糖的分子量、溶液中游离氨基的浓度以及壳聚糖骨架上金属的密度对其与DNA的作用模式也有不同程度的影响。
第三章 纳米壳聚糖水杨醛希夫碱金属配合物的合成及其与DNA作用的研究
将水溶性纳米壳聚糖COS3(19000)和)和COS4(4000)经水杨醛改性后与Cu(Ⅱ)、Ni(Ⅱ)、Zn(Ⅱ)、Co(Ⅱ)四种金属配位合成了一系列壳聚糖水杨醛希夫碱金属配合物。并对配合物的结构和组成进行了表征。
采用紫外光谱、荧光光谱和琼脂糖凝胶电泳三种分析手段首次研究了COS
一Met系列壳聚糖金属配合物与DNA的作用，并提出了可能的作用机理和模式。
结果表明此类壳聚糖金属配合物与DNA作用时存在静电结合和嵌入两种作用模
式。由于COS分子量的降低和配合物平面性增强使其与DNA的作用程度明显增
强，同时，考察了纳米壳聚糖的分子量、溶液中游离氨基的浓度以及壳聚糖骨架
上金属的密度对COS一Sal一Met与DNA的作用模式和作用程度的影响。
第四章纳米壳聚糖卜毗咤醛希夫碱金属配合物的合成及其与DNA作用的研究
首次合成了一系列纳米壳聚糖卜毗陡醛希夫碱金属(Cu、Ni、Zn、Co)配
合物，并对配合物的结构和组成进行了表征。
采用紫外光谱，荧光光谱和琼脂糖凝胶电泳三种分析手段首次研究了COS
一P一Mct系列壳聚糖金属配合物与DNA的作用。此类壳聚糖金属配合物与
DNA作用时存在静电结合模式。同时DNA也有不同程度的降解，其机理还有其
他作用模式有待于进一步研究。
Chitin/chitosan and their derivatives have been various applied in the pharmaceutical field, due to theirs good physical chemistry property and pharmaceutical function. In recently, chitin and chitosan of low molecular weight have been significant noted because of their many encouraging results and specific features. In this review, it is focus on the achievement in the field of pharmaceutical application, such as pharmaceutical function and biological activity of chitin/ chitosan and theirs derivatives, the application as drug carrier material and as gene delivery vector in gene therapy.
Chitosan was depolymerized with oxidation method, which afforded nano-chitosan of low molecular weights (COS Mw=20 000, Mw=19 000, 4 000). Nano-chitosan metal complexes (COS-Met), Nano-chitosan salicylaldehyde Schiff base metal complexes (COS-Sal-Met) and a series of novel Nano-chitosanm a-pyridine-carboxaldehyde Schiff base metal complexes (COS-P-Met) with Cu (II), Ni( II ), Zn( II), Co( II) were synthesized. The nano-chitosans and their complexes were characterized by means of gel permeation chromatography (GPC), IR spectra, Elemental analysis and ICP. The interaction of DNA with these nano-chitosan metal complexes was studied by UV/Vis, fluorescent probe technicality and electrophoresis analysis at the first time. The possible interaction mechanism and mode were discussed.
The results indicate that interaction between the nano-chitosan metal complex (COS-Met), Nano-chitosan salicylaldehyde Schiff base metal complexes (COS-Sal-Met) with DNA has two modes. Firstly, the free amine positive charged of nano-chitosan backbone electrostatic bind to negative charged phosphate backbone at the periphery of the double helix DNA, then double helix DNA contracted at axial and its conformation was changed. Secondly, a portion of ligands intercalated between the base pair of the DNA duplex helix cause the secondary structure of the DNA destroyed. Furthermore, the increasing water solubility and the plenary complex structure of
(COS-Sal-Met) improved theirs reaction activity. The nano-chitosan a-pyridine-carboxaldehyde Schiff base metal complexes (COS-P-Met) interacting with DNA has electrostatic binding mode being observed. It should be further studied to determine whether there are other modes existed. At the same time, the influence on their interaction degree and mode of three kinds of nano-chitosan metal complexes chemical structure, such as the molecular weights of nano-chitosan, the content of free amine and metal of nano-chitosan metal complexes, were determined.
引文
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