基于改性聚乙烯亚胺的基因药物传递体系研究
发布时间：2019-04-28 11:14
【摘要】：针对目前基因及药物传递体系前沿的研究方向,我们将改性聚乙烯亚胺(PEI)和超分子组装技术结合,为构建新型的基因传递体系和基因、药物控释涂层提供新途径。 本研究首先成功合成了具有pH刺激响应电荷逆转的大分子CPEI,其在中性条件下表面电位在-20mV左右,呈负电性,但在酸性条件下,呈现出电荷逆转的特点带上正电,表面电位在+2.0mV。随着在酸性环境中时间的增加,其正电性也由于侧基的水解而增强。以此大分子为组分,与预组装得到的PEI-DNA复合粒子进行层层自组装,构建得到负载有PEI-DNA复合粒子的多层膜,12双层的膜厚接近120nm。进一步的研究表明,该多层膜呈现出了CPEI所带来的电荷逆转的特点,能够对pH发生刺激响应,在pH小于6.0的情况下释放得到含有DNA的复合粒子。这一具有pH响应功能的基因薄膜涂层为原位释放、转染细胞提供了新的可能。 研究进一步制备了二茂铁修饰的PEI (BPEI-Fc),利用二茂铁的疏水性诱导形成胶束,其临界胶束浓度为0.046mg/mL。该胶束成功负载了疏水性的小分子药物阿霉素,包覆量为7.7%。载有阿霉素的胶束与DNA成功复合形成药物基因双传递的预组装体,并与磺化葡聚糖进行了层层自组装,构建了具有药物基因双传递功能的多层膜。该种多层膜50双层能够负载阿霉素6.951μg/cm2,并能在双氧水刺激下释放出接近90%的阿霉素。在随后的细胞试验中发现,载入多层膜内的阿霉素确实能够抑制肝癌细胞的生长,50双层的多层膜72h小时相对TCPS的细胞活性不到20%。绿色荧光蛋白质粒DNA的转染实验也证实,多层膜能够实现HEK293细胞和肝癌细胞的转染和蛋白质的表达。这一具备药物和基因双传递功能的薄膜涂层为解决癌症抗药性问题提供了新的途径。 最后,利用二茂铁与β-环糊精的主客体嵌套作用,将已合成的二茂铁修饰的PEI,与β-环糊精充分复合形成粒径在169nm的粒子。将粒子与DNA复合后,构建了β-环糊精辅助传递DNA的液相传递粒子,考察了不同N/P值下的复合粒子的理化特性及复合规律。在N/P值为1时,DNA尚未完全与BPEI-Fc-CD复合,在N/P值为2时,DNA才完全被BPEI-Fc-CD复合到粒子中。当N/P值大于等于7时,DNA才被完全的包覆在了粒子内部。稳定性实验表明,粒子在体外细胞培养环境中相对稳定,没有发生明显的聚集。在转染实验中发现,该种液相的传递基因的复合粒子,在环糊精的辅助作用下,确实能够实现相对于传统PEI而言更高的转染率和更好的细胞相容性,其基因转染效率超过18%。这一结果为构建理想的基因传递体系提供了新的可能。
[Abstract]:According to the current research direction of gene and drug delivery system, we combine modified polyethyleneimine (PEI) with supramolecular assembly technology to provide a new way to construct new gene delivery system and gene, drug controlled release coating. In this study, we successfully synthesized the macromolecule CPEI, with charge reversal in response to pH stimulation. The surface potential is about-20mV in neutral condition, which is negative, but in acidic condition, it shows the characteristic of charge reversal on the positive charge. The surface potential is 2.0mV. With the increase of time in acidic environment, the positive electrical properties of these compounds were enhanced by the hydrolysis of side groups. With the macromolecule as the component, the PEI-DNA composite particles were assembled by self-assembly layer by layer, and the multilayer films loaded with PEI-DNA composite particles were constructed. The thickness of the 12 bilayers was close to 120 nm. Further studies show that the multilayer film has the characteristics of charge reversal caused by CPEI, which can stimulate pH and release DNA-containing composite particles when pH is less than 6.0.The composite particles containing DNA are released. This pH-responsive gene film coating provides a new possibility for in-situ release of transfected cells. Ferrocene modified PEI (BPEI-Fc) was further prepared. The micelle was induced by ferrocene hydrophobicity and its critical micelle concentration was 0.046 mg / L. The micelle was successfully loaded with hydrophobic small molecular drug adriamycin (7.7%). The micelles containing doxorubicin were successfully combined with DNA to form a preassembly with double transfer of drug gene and self-assembled layer by layer with sulfonated dextran to construct a multilayer membrane with double transfer function of drug gene. The multilayer 50 bilayers can load 6.951 渭 g / cm2, doxorubicin and release nearly 90% doxorubicin under hydrogen peroxide stimulation. In subsequent cell tests it was found that doxorubicin loaded in multilayered membranes did inhibit the growth of hepatoma cells and that 50 bilayer multilayer membranes showed less than 20% of TCPS activity at 72 h. The transfection experiment of green fluorescent protein particle DNA also confirmed that the multilayer membrane could realize the transfection and protein expression of HEK293 cells and hepatoma cells. This thin film coating, which has both drug and gene transfer functions, provides a new way to solve the problem of cancer resistance. Finally, the ferrocene modified PEI, and 尾-cyclodextrin were synthesized by the host-guest nesting of ferrocene and 尾-cyclodextrin to form 169nm particles. The liquid phase transport particles assisted by 尾-cyclodextrin (尾-cyclodextrin) were constructed after the particles were mixed with DNA. The physical and chemical properties of the composite particles with different N / P ratios were investigated. When the N / P value is 1, DNA has not been completely combined with BPEI-Fc-CD, and when the N / P value is 2, the DNA is completely compounded into the particles by BPEI-Fc-CD. When the N P value is greater than or equal to 7, the DNA is completely coated inside the particle. The stability test showed that the particles were relatively stable in the cell culture environment in vitro, and there was no obvious aggregation of the particles. In the transfection experiment, it was found that the complex particles of this kind of liquid phase transfer gene, assisted by cyclodextrin, could achieve higher transfection rate and better cell compatibility compared with the traditional PEI, and the gene transfection efficiency was more than 18%. These results provide a new possibility for constructing an ideal gene transfer system.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
本文编号：2467584
[Abstract]:According to the current research direction of gene and drug delivery system, we combine modified polyethyleneimine (PEI) with supramolecular assembly technology to provide a new way to construct new gene delivery system and gene, drug controlled release coating. In this study, we successfully synthesized the macromolecule CPEI, with charge reversal in response to pH stimulation. The surface potential is about-20mV in neutral condition, which is negative, but in acidic condition, it shows the characteristic of charge reversal on the positive charge. The surface potential is 2.0mV. With the increase of time in acidic environment, the positive electrical properties of these compounds were enhanced by the hydrolysis of side groups. With the macromolecule as the component, the PEI-DNA composite particles were assembled by self-assembly layer by layer, and the multilayer films loaded with PEI-DNA composite particles were constructed. The thickness of the 12 bilayers was close to 120 nm. Further studies show that the multilayer film has the characteristics of charge reversal caused by CPEI, which can stimulate pH and release DNA-containing composite particles when pH is less than 6.0.The composite particles containing DNA are released. This pH-responsive gene film coating provides a new possibility for in-situ release of transfected cells. Ferrocene modified PEI (BPEI-Fc) was further prepared. The micelle was induced by ferrocene hydrophobicity and its critical micelle concentration was 0.046 mg / L. The micelle was successfully loaded with hydrophobic small molecular drug adriamycin (7.7%). The micelles containing doxorubicin were successfully combined with DNA to form a preassembly with double transfer of drug gene and self-assembled layer by layer with sulfonated dextran to construct a multilayer membrane with double transfer function of drug gene. The multilayer 50 bilayers can load 6.951 渭 g / cm2, doxorubicin and release nearly 90% doxorubicin under hydrogen peroxide stimulation. In subsequent cell tests it was found that doxorubicin loaded in multilayered membranes did inhibit the growth of hepatoma cells and that 50 bilayer multilayer membranes showed less than 20% of TCPS activity at 72 h. The transfection experiment of green fluorescent protein particle DNA also confirmed that the multilayer membrane could realize the transfection and protein expression of HEK293 cells and hepatoma cells. This thin film coating, which has both drug and gene transfer functions, provides a new way to solve the problem of cancer resistance. Finally, the ferrocene modified PEI, and 尾-cyclodextrin were synthesized by the host-guest nesting of ferrocene and 尾-cyclodextrin to form 169nm particles. The liquid phase transport particles assisted by 尾-cyclodextrin (尾-cyclodextrin) were constructed after the particles were mixed with DNA. The physical and chemical properties of the composite particles with different N / P ratios were investigated. When the N / P value is 1, DNA has not been completely combined with BPEI-Fc-CD, and when the N / P value is 2, the DNA is completely compounded into the particles by BPEI-Fc-CD. When the N P value is greater than or equal to 7, the DNA is completely coated inside the particle. The stability test showed that the particles were relatively stable in the cell culture environment in vitro, and there was no obvious aggregation of the particles. In the transfection experiment, it was found that the complex particles of this kind of liquid phase transfer gene, assisted by cyclodextrin, could achieve higher transfection rate and better cell compatibility compared with the traditional PEI, and the gene transfection efficiency was more than 18%. These results provide a new possibility for constructing an ideal gene transfer system.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R341
【参考文献】
相关期刊论文 前1条
1 吴涛,张凤鱼;非病毒型基因传递系统[J];沈阳药科大学学报;2001年04期
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