壳聚糖衍生物非病毒基因载体的研究
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摘要
壳聚糖是一种天然的阳离子多糖,具有良好的生物相容性,生物降解性,低免疫原性以及无毒性的优点。壳聚糖通过静电作用可以与DNA形成聚电解质复合物,保护DNA免受核酸酶的降解,从而促使DNA顺利的进入细胞,因此壳聚糖作为非病毒基因载体具有广泛的应用前景。
本文制备了两种新型的壳聚糖非病毒载体,精氨酸修饰的壳聚糖(Arg-CS)和N-亚甲基磷酸化壳聚糖(NMPCS),探讨其物理化学性质及其与DNA之间的相互作用及基因转染行为,对壳聚糖载基因纳米粒子荧光标记条件进行了优化,探讨了精氨酸修饰壳聚糖载基因血管支架体内局部释放基因的可行性,具体内容主要包括以下几个方面:
1、精氨酸修饰的壳聚糖的制备及其与DNA相互作用的研究。通过红外光谱和核磁共振图谱分析,可以确认精氨酸已经共价连接到壳聚糖上。圆二色谱结果表明DNA与壳聚糖作用形成纳米尺寸的复合物后仍然保持着典型的B型构象。用透射电镜,原子力显微镜和光子相关光谱检测复合物的表面形态和尺寸大小分布,结果表明,精氨酸修饰的壳聚糖/DNA纳米复合物(N/P=2:1)呈现不规则的球形,复合物的直径分布大多在80～150nm之间,有利于基因转染。
2、精氨酸修饰的壳聚糖介导的基因转染研究。精氨酸修饰的壳聚糖介导荧光素酶质粒的表达较未修饰的壳聚糖介导的荧光素酶质粒的表达提高了大约100倍,在整个实验浓度范围内,精氨酸修饰的壳聚糖及其与DNA的复合物对HeLa细胞的毒性非常小。
3、N-亚甲基磷酸化壳聚糖的制备及其与DNA相互作用的研究。以红外光谱(FTIR)分析表明改性后的壳聚糖的特征基团有显著变化;13C NMR检测进一步证明了亚甲基磷酸基团的引入。通过复凝聚的方法,制备了纳米尺寸的NMPCS/DNA聚电解质复合物。考察了共聚物与DNA的相互作用情况,复合物中DNA的构象变化和复合物的形态和粒径。圆二色谱结果表明DNA与壳聚糖作用形成纳米尺寸的复合物后仍然保持着典型的B型构象。透射电镜,原子力显微镜检测结果表明,不同N/P下的N-亚甲基磷酸化修饰的壳聚糖/DNA纳米复合物呈现较规则的球形,且在高电荷比时可形成尺寸较小的纳米尺度颗粒。
4、以人宫颈癌细胞HeLa为宿主细胞,尝试了以NMPCS为载体介导含荧光素酶的pGL3质粒的体外转染,重点研究了pH值和N/P比对转染效率的影响。实验结果表明,载体可有效将质粒转染HeLa细胞,获得的最佳转染效率远高于裸DNA和CS,与PEI相当。基于磷酸基团对钙离子有良好的鳌合能力制备了聚合物盐型NMPCS-Ca载体,并用于介导基因转染,其转染过程类似于传统的P-Ca载体,并认为Ca~(2+)离子通道促进了转染过程。
5、壳聚糖载基因纳米粒子荧光标记的优化研究。考察了FITC与壳聚糖反应的主要影响因素,观察了荧光标记对纳米粒子表征和转染效率的影响,建立一种快速、稳定并且适宜壳聚糖纳米载体示踪的荧光标记方法。壳聚糖纳米粒子表征和转染效率实验显示最佳的壳聚糖标记条件是反应物壳聚糖与FITC的添加比例为25:1,在pH值7.5室温25℃反应4小时。
6、精氨酸修饰壳聚糖载基因支架血管内基因转运的研究。细胞转染实验显示,支架表面细胞及支架邻近细胞大量转染,远离支架细胞未见转染。动物实验结果显示,支架植入部位荧光素酶高表达,同时有2只动物肝脏标本有微量表达,其他部位未见表达。精氨酸修饰壳聚糖质粒DNA支架有望成为一种新型有效的血管内基因转运体系。
Chitosan is a naturally occurring cationic polysaccharide with good biocompatibility, biodegradation, low immunogenicity and non-cytotoxicity. Chitosan is considered to be a good candidate for non-viral vector, since cationically charged chitosan can efficiently condense negatively charged DNA to form polyelectrolyte complexes via electrostatic interaction, facilitating the transport of therapeutic genes across the cell membrane.
In this thesis, two novel non-viral vectors, arginine-modified chitosan (Arg-CS) and N-methylene phosphonic chitosan (NMPCS), were developed. The physicochemical properties of the vectors,the interactions between the vectors and plasmid DNA and gene tranfection activities were investigated. The parameters of labeling chitosan-DNA nanoparticles with fluorescein isothiocyanate were optimized. The potential application of Arg-CS/DNA nanoprticles (ACDNPs) coated stent in endovascular gene delivery was studied. The work mainly focus on the following parts:
1. Synthesis of Arg-CS and interactions between Arg-CS and DNA.. The formation of Arg-CS and the properties of Arg-CS/DNA complexes were investigated. FTIR and 13C NMR spectra results showed that arginine was chemically coupled to chitosan to form arginine-modified chitosan conjugates. CD spectra indicated that the interaction of DNA with chitosan merely caused a slight perturbation of DNA bonds and DNA still remained B-conformation within the complexes. TEM, AFM and DLS results revealed that the complexes were nearly spheres with mean diameter about 80-150nm, which is very suitable for gene delivery.
2. Gene transfection mediated by Arg-CS. Luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan. MTT assay indicated that Arg-CS was a safe non viral vector.
3. Synthesis of NMPCS and interactions between NMPCS and DNA. FTIR and 13C NMR spectra were used to study the structure of NMPCS. NMPCS/DNA complexes were obtained using a complex coacervation process, characterized by agarose gel electrophoresis retardation assay for their stabilities, atomic force microscopy (AFM), transmission electron microscopy (TEM) and laser size analysizer observation for morphology and size, results showed that at charge ratio 2:1 or above, DNA could be completely entrapped and spherical complexes with mean size of 80~210nm were formed.
4. Gene transfection mediated by NMPCS. The results indicated that the transfection efficiencies were strongly dependent on the charge ratio and pH value of culture medium, for HeLa cells, the highest efficiency obtained at ratio of 4:1 and pH 6.2 was greatly higher than that from CS/DNA complexes or naked DNA and approximate to that from PEI/DNA complexes. Polymer-inorganic salt type NMPCS-Ca vector showed some similarities with traditional Ca-P, giving high efficiencies probably owe to positive role of Ca2+ ion channel.
5. Optimization of the assay for labeling chitosan-DNA nanoparticles with fluorescein isothiocyanate. To achieve the appropriate nanoparticle size and cell transfection efficacy, the FCNP should be prepared with the FITC-chitosan labeled by incubating the FITC and chitosan in a pH 7.5 solution at 25℃for 4 hours with the molar ratio of chitosan to FITC being 25:1.
6. Study on Arg-CS/DNA nanoprticles (ACDNPs) coated stent in endovascular gene delivery. In A10 cell culture, the ACDNP-stents with plasmid pIRES-EGFP induced high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, luciferase activity was observed only in the region of the artery in contact with the ACDNP-stents(high expression) but not in adjacent arterial segments or at the distal organs(2 liver samples showed low luciferase activity). Arg-CS/DNA nanoprticles (ACDNPs) coated stents demonstrate great potential as a novel and effective endovascular gene delivery system.
本文制备了两种新型的壳聚糖非病毒载体,精氨酸修饰的壳聚糖(Arg-CS)和N-亚甲基磷酸化壳聚糖(NMPCS),探讨其物理化学性质及其与DNA之间的相互作用及基因转染行为,对壳聚糖载基因纳米粒子荧光标记条件进行了优化,探讨了精氨酸修饰壳聚糖载基因血管支架体内局部释放基因的可行性,具体内容主要包括以下几个方面:
1、精氨酸修饰的壳聚糖的制备及其与DNA相互作用的研究。通过红外光谱和核磁共振图谱分析,可以确认精氨酸已经共价连接到壳聚糖上。圆二色谱结果表明DNA与壳聚糖作用形成纳米尺寸的复合物后仍然保持着典型的B型构象。用透射电镜,原子力显微镜和光子相关光谱检测复合物的表面形态和尺寸大小分布,结果表明,精氨酸修饰的壳聚糖/DNA纳米复合物(N/P=2:1)呈现不规则的球形,复合物的直径分布大多在80～150nm之间,有利于基因转染。
2、精氨酸修饰的壳聚糖介导的基因转染研究。精氨酸修饰的壳聚糖介导荧光素酶质粒的表达较未修饰的壳聚糖介导的荧光素酶质粒的表达提高了大约100倍,在整个实验浓度范围内,精氨酸修饰的壳聚糖及其与DNA的复合物对HeLa细胞的毒性非常小。
3、N-亚甲基磷酸化壳聚糖的制备及其与DNA相互作用的研究。以红外光谱(FTIR)分析表明改性后的壳聚糖的特征基团有显著变化;13C NMR检测进一步证明了亚甲基磷酸基团的引入。通过复凝聚的方法,制备了纳米尺寸的NMPCS/DNA聚电解质复合物。考察了共聚物与DNA的相互作用情况,复合物中DNA的构象变化和复合物的形态和粒径。圆二色谱结果表明DNA与壳聚糖作用形成纳米尺寸的复合物后仍然保持着典型的B型构象。透射电镜,原子力显微镜检测结果表明,不同N/P下的N-亚甲基磷酸化修饰的壳聚糖/DNA纳米复合物呈现较规则的球形,且在高电荷比时可形成尺寸较小的纳米尺度颗粒。
4、以人宫颈癌细胞HeLa为宿主细胞,尝试了以NMPCS为载体介导含荧光素酶的pGL3质粒的体外转染,重点研究了pH值和N/P比对转染效率的影响。实验结果表明,载体可有效将质粒转染HeLa细胞,获得的最佳转染效率远高于裸DNA和CS,与PEI相当。基于磷酸基团对钙离子有良好的鳌合能力制备了聚合物盐型NMPCS-Ca载体,并用于介导基因转染,其转染过程类似于传统的P-Ca载体,并认为Ca~(2+)离子通道促进了转染过程。
5、壳聚糖载基因纳米粒子荧光标记的优化研究。考察了FITC与壳聚糖反应的主要影响因素,观察了荧光标记对纳米粒子表征和转染效率的影响,建立一种快速、稳定并且适宜壳聚糖纳米载体示踪的荧光标记方法。壳聚糖纳米粒子表征和转染效率实验显示最佳的壳聚糖标记条件是反应物壳聚糖与FITC的添加比例为25:1,在pH值7.5室温25℃反应4小时。
6、精氨酸修饰壳聚糖载基因支架血管内基因转运的研究。细胞转染实验显示,支架表面细胞及支架邻近细胞大量转染,远离支架细胞未见转染。动物实验结果显示,支架植入部位荧光素酶高表达,同时有2只动物肝脏标本有微量表达,其他部位未见表达。精氨酸修饰壳聚糖质粒DNA支架有望成为一种新型有效的血管内基因转运体系。
Chitosan is a naturally occurring cationic polysaccharide with good biocompatibility, biodegradation, low immunogenicity and non-cytotoxicity. Chitosan is considered to be a good candidate for non-viral vector, since cationically charged chitosan can efficiently condense negatively charged DNA to form polyelectrolyte complexes via electrostatic interaction, facilitating the transport of therapeutic genes across the cell membrane.
In this thesis, two novel non-viral vectors, arginine-modified chitosan (Arg-CS) and N-methylene phosphonic chitosan (NMPCS), were developed. The physicochemical properties of the vectors,the interactions between the vectors and plasmid DNA and gene tranfection activities were investigated. The parameters of labeling chitosan-DNA nanoparticles with fluorescein isothiocyanate were optimized. The potential application of Arg-CS/DNA nanoprticles (ACDNPs) coated stent in endovascular gene delivery was studied. The work mainly focus on the following parts:
1. Synthesis of Arg-CS and interactions between Arg-CS and DNA.. The formation of Arg-CS and the properties of Arg-CS/DNA complexes were investigated. FTIR and 13C NMR spectra results showed that arginine was chemically coupled to chitosan to form arginine-modified chitosan conjugates. CD spectra indicated that the interaction of DNA with chitosan merely caused a slight perturbation of DNA bonds and DNA still remained B-conformation within the complexes. TEM, AFM and DLS results revealed that the complexes were nearly spheres with mean diameter about 80-150nm, which is very suitable for gene delivery.
2. Gene transfection mediated by Arg-CS. Luciferase expression mediated by Arg-CS was greatly enhanced to about 100 folds compared with the luciferase expression mediated by chitosan. MTT assay indicated that Arg-CS was a safe non viral vector.
3. Synthesis of NMPCS and interactions between NMPCS and DNA. FTIR and 13C NMR spectra were used to study the structure of NMPCS. NMPCS/DNA complexes were obtained using a complex coacervation process, characterized by agarose gel electrophoresis retardation assay for their stabilities, atomic force microscopy (AFM), transmission electron microscopy (TEM) and laser size analysizer observation for morphology and size, results showed that at charge ratio 2:1 or above, DNA could be completely entrapped and spherical complexes with mean size of 80~210nm were formed.
4. Gene transfection mediated by NMPCS. The results indicated that the transfection efficiencies were strongly dependent on the charge ratio and pH value of culture medium, for HeLa cells, the highest efficiency obtained at ratio of 4:1 and pH 6.2 was greatly higher than that from CS/DNA complexes or naked DNA and approximate to that from PEI/DNA complexes. Polymer-inorganic salt type NMPCS-Ca vector showed some similarities with traditional Ca-P, giving high efficiencies probably owe to positive role of Ca2+ ion channel.
5. Optimization of the assay for labeling chitosan-DNA nanoparticles with fluorescein isothiocyanate. To achieve the appropriate nanoparticle size and cell transfection efficacy, the FCNP should be prepared with the FITC-chitosan labeled by incubating the FITC and chitosan in a pH 7.5 solution at 25℃for 4 hours with the molar ratio of chitosan to FITC being 25:1.
6. Study on Arg-CS/DNA nanoprticles (ACDNPs) coated stent in endovascular gene delivery. In A10 cell culture, the ACDNP-stents with plasmid pIRES-EGFP induced high level of GFP expression in cells grown on the stent surface and along the adjacent area. In animal studies, luciferase activity was observed only in the region of the artery in contact with the ACDNP-stents(high expression) but not in adjacent arterial segments or at the distal organs(2 liver samples showed low luciferase activity). Arg-CS/DNA nanoprticles (ACDNPs) coated stents demonstrate great potential as a novel and effective endovascular gene delivery system.
引文
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