新型壳聚糖衍生物的合成及其基因转运研究
发布时间：2018-11-21 10:05
【摘要】：基因治疗需要解决的首要问题是开发安全、高效的基因载体。壳聚糖(CS)作为基因载体具有生物相容性好、安全性高等优点,但不经改性修饰的CS基因转运效率低;而CS的改性修饰又因其在非水介质中的难溶性,受到诸多限制。1-丁基-3-甲基咪唑醋酸盐([BMIM]Ac)离子液体可以溶解CS,解决了制备新型壳聚糖衍生物的溶解性限制难题,并有利于促进反应进行、提高取代度。本论文采用[BMIM]Ac溶剂,合成了壳聚糖接枝聚乙烯亚胺(PEI)、壳聚糖接枝单甲氧基聚乙二醇胺(mPEG-NH2)和PEI、双重刺激响应壳聚糖接枝mPEG-NH2和PEI衍生物,并研究了它们的基因转运性能。以羰基二咪唑(CDI)为选择性活化试剂,[BMIM]Ac为均相溶剂,通过两步亲核取代反应对CS进行PEI高效接枝,得到一系列CS-g-PEI-x脲类接枝聚合物,反应速度快、产物取代度高。采用FTIR、1HNMR、13C-CP/MAS NMR、GPC等手段对产物结构进行了表征。酸碱滴定、琼脂糖凝胶电泳和激光粒度仪测试结果表明,随着PEI接枝率的增加,CS-g-PEI-x对DNA的延滞能力和压缩能力增强。在Hep-2细胞中的基因转运结果表明,PEI接枝率为4.5%时,CS-g-PEI具有最佳的基因转运效率和较低的细胞毒性。采用上述方法,以mPEG-NH2和PEI对CS进行修饰,得到CS-g-mPEG-g-PEI-x接枝聚合物。FTIR、1HNMR、UV、GPC等测试结果表明,mPEG-NH2和PEI接枝到CS分子链上。酸碱滴定和琼脂糖凝胶电泳表明,PEI接枝率接近时,提高mPEG-NH2接枝率对质子缓冲能力影响不显著,但DNA延滞能力减弱。氮磷比(N/P)为6的CS-g-mPEG-g-PEI-x/DNA复合物能够有效抵抗0.10μg/μL肝素钠和2.0 U核酸酶的破坏。10%血清条件下Hep-2细胞中的基因转运结果表明,CS-g-mPEG-g-PEI聚合物细胞毒性低,mPEG-NH2接枝率为2.9%时基因转运效率最好。从壳寡糖出发,合成了酸敏感酰腙键和还原敏感二硫键交联双重刺激响应壳聚糖(SRCS):采用前述方法,对SRCS进行mPEG-NH2和PEI共价接枝修饰,得到SRCS-g-mPEG-g-PEI聚合物。利用1HNMR、13C-CP/MAS NMR、FTIR、UV和GPC等手段对产物结构进行了表征。SRCS-g-mPEG-g-PEI能够有效压缩DNA,形成的复合物(N/P≥10)在0.10μg/μL肝素钠、2.0 U核酸酶及25%血清中能够稳定存在,在pH=5.0的酸环境或10 mM DTT的还原环境中可以响应释放DNA。Hep-2细胞中的基因转运结果表明,SRCS-g-mPEG-g-PEI在10%血清条件下具有良好的基因转运性能,有望成为安全、高效的壳聚糖基非病毒基因载体。
[Abstract]:The most important problem for gene therapy is to develop a safe and efficient gene vector. Chitosan (CS) as a gene vector has the advantages of good biocompatibility and high safety, but the CS gene transport efficiency without modification is low. However, the modification of CS is limited by its insolubility in non-aqueous medium. 1 Ding Ji 3 methyl imidazole acetate ([BMIM] Ac) ionic liquid can dissolve CS,. It solves the problem of solubility limitation in the preparation of new chitosan derivatives, and helps to promote the reaction and improve the degree of substitution. In this paper, chitosan grafted polyethylene imine (PEI), chitosan onto monomethoxy polyethylene glycol amine (mPEG-NH2) and PEI, double stimulus responsive chitosan graft mPEG-NH2 and PEI derivatives were synthesized by using [BMIM] Ac solvent. Their gene transport properties were also studied. Using carbonyl diimidazole (CDI) as selective activation reagent and [BMIM] Ac as homogeneous solvent, a series of CS-g-PEI-x ureas grafted polymers were obtained by two steps nucleophilic substitution reaction to PEI. The degree of substitution of the product is high. The structure of the product was characterized by FTIR,1HNMR,13C-CP/MAS NMR,GPC. The results of acid-base titration, agarose gel electrophoresis and laser particle size analyzer showed that with the increase of PEI grafting rate, the CS-g-PEI-x 's tardiness and compression ability to DNA were enhanced. The results of gene transport in Hep-2 cells showed that CS-g-PEI had the best gene transport efficiency and low cytotoxicity when the grafting rate of PEI was 4.5. CS was modified by mPEG-NH2 and PEI, and CS-g-mPEG-g-PEI-x grafted polymer was obtained by the method mentioned above. The results of FTIR,1HNMR,UV,GPC and FTIR,1HNMR,UV,GPC showed that mPEG-NH2 and PEI were grafted onto CS molecular chain. Acid-base titration and agarose gel electrophoresis showed that when the grafting ratio of PEI was close to that of mPEG-NH2, the proton buffering capacity was not significantly affected by increasing the graft ratio of mPEG-NH2, but the tardiness ability of DNA was weakened. The CS-g-mPEG-g-PEI-x/DNA complex with N / P ratio of 6 could effectively resist the destruction of 0.10 渭 g / 渭 L heparin sodium and 2.0 U nuclease. The cytotoxicity of CS-g-mPEG-g-PEI polymer was low, and the efficiency of gene transport was the best when the grafting rate of mPEG-NH2 was 2.9. The acid-sensitive acylhydrazone bond and reduction-sensitive disulfide bond crosslinking double stimulatory response chitosan (SRCS): were synthesized from chitosan oligosaccharide. The SRCS was covalently modified by mPEG-NH2 and PEI to obtain SRCS-g-mPEG-g-PEI polymer by the method mentioned above. The structure of the product was characterized by 1H-NMR-13C-CP-MAS NMR,FTIR,UV and GPC. SRCS-g-mPEG-g-PEI can effectively compress the complex formed by DNA, (N / P 鈮，
本文编号：2346653
[Abstract]:The most important problem for gene therapy is to develop a safe and efficient gene vector. Chitosan (CS) as a gene vector has the advantages of good biocompatibility and high safety, but the CS gene transport efficiency without modification is low. However, the modification of CS is limited by its insolubility in non-aqueous medium. 1 Ding Ji 3 methyl imidazole acetate ([BMIM] Ac) ionic liquid can dissolve CS,. It solves the problem of solubility limitation in the preparation of new chitosan derivatives, and helps to promote the reaction and improve the degree of substitution. In this paper, chitosan grafted polyethylene imine (PEI), chitosan onto monomethoxy polyethylene glycol amine (mPEG-NH2) and PEI, double stimulus responsive chitosan graft mPEG-NH2 and PEI derivatives were synthesized by using [BMIM] Ac solvent. Their gene transport properties were also studied. Using carbonyl diimidazole (CDI) as selective activation reagent and [BMIM] Ac as homogeneous solvent, a series of CS-g-PEI-x ureas grafted polymers were obtained by two steps nucleophilic substitution reaction to PEI. The degree of substitution of the product is high. The structure of the product was characterized by FTIR,1HNMR,13C-CP/MAS NMR,GPC. The results of acid-base titration, agarose gel electrophoresis and laser particle size analyzer showed that with the increase of PEI grafting rate, the CS-g-PEI-x 's tardiness and compression ability to DNA were enhanced. The results of gene transport in Hep-2 cells showed that CS-g-PEI had the best gene transport efficiency and low cytotoxicity when the grafting rate of PEI was 4.5. CS was modified by mPEG-NH2 and PEI, and CS-g-mPEG-g-PEI-x grafted polymer was obtained by the method mentioned above. The results of FTIR,1HNMR,UV,GPC and FTIR,1HNMR,UV,GPC showed that mPEG-NH2 and PEI were grafted onto CS molecular chain. Acid-base titration and agarose gel electrophoresis showed that when the grafting ratio of PEI was close to that of mPEG-NH2, the proton buffering capacity was not significantly affected by increasing the graft ratio of mPEG-NH2, but the tardiness ability of DNA was weakened. The CS-g-mPEG-g-PEI-x/DNA complex with N / P ratio of 6 could effectively resist the destruction of 0.10 渭 g / 渭 L heparin sodium and 2.0 U nuclease. The cytotoxicity of CS-g-mPEG-g-PEI polymer was low, and the efficiency of gene transport was the best when the grafting rate of mPEG-NH2 was 2.9. The acid-sensitive acylhydrazone bond and reduction-sensitive disulfide bond crosslinking double stimulatory response chitosan (SRCS): were synthesized from chitosan oligosaccharide. The SRCS was covalently modified by mPEG-NH2 and PEI to obtain SRCS-g-mPEG-g-PEI polymer by the method mentioned above. The structure of the product was characterized by 1H-NMR-13C-CP-MAS NMR,FTIR,UV and GPC. SRCS-g-mPEG-g-PEI can effectively compress the complex formed by DNA, (N / P 鈮，
本文编号：2346653
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