新型殼聚糖衍生物的合成及其基因轉(zhuǎn)運研究
發(fā)布時間：2018-11-21 10:05
【摘要】：基因治療需要解決的首要問題是開發(fā)安全、高效的基因載體。殼聚糖(CS)作為基因載體具有生物相容性好、安全性高等優(yōu)點,但不經(jīng)改性修飾的CS基因轉(zhuǎn)運效率低;而CS的改性修飾又因其在非水介質(zhì)中的難溶性,受到諸多限制。1-丁基-3-甲基咪唑醋酸鹽([BMIM]Ac)離子液體可以溶解CS,解決了制備新型殼聚糖衍生物的溶解性限制難題,并有利于促進反應(yīng)進行、提高取代度。本論文采用[BMIM]Ac溶劑,合成了殼聚糖接枝聚乙烯亞胺(PEI)、殼聚糖接枝單甲氧基聚乙二醇胺(mPEG-NH2)和PEI、雙重刺激響應(yīng)殼聚糖接枝mPEG-NH2和PEI衍生物,并研究了它們的基因轉(zhuǎn)運性能。以羰基二咪唑(CDI)為選擇性活化試劑,[BMIM]Ac為均相溶劑,通過兩步親核取代反應(yīng)對CS進行PEI高效接枝,得到一系列CS-g-PEI-x脲類接枝聚合物,反應(yīng)速度快、產(chǎn)物取代度高。采用FTIR、1HNMR、13C-CP/MAS NMR、GPC等手段對產(chǎn)物結(jié)構(gòu)進行了表征。酸堿滴定、瓊脂糖凝膠電泳和激光粒度儀測試結(jié)果表明,隨著PEI接枝率的增加,CS-g-PEI-x對DNA的延滯能力和壓縮能力增強。在Hep-2細胞中的基因轉(zhuǎn)運結(jié)果表明,PEI接枝率為4.5%時,CS-g-PEI具有最佳的基因轉(zhuǎn)運效率和較低的細胞毒性。采用上述方法,以mPEG-NH2和PEI對CS進行修飾,得到CS-g-mPEG-g-PEI-x接枝聚合物。FTIR、1HNMR、UV、GPC等測試結(jié)果表明,mPEG-NH2和PEI接枝到CS分子鏈上。酸堿滴定和瓊脂糖凝膠電泳表明,PEI接枝率接近時,提高mPEG-NH2接枝率對質(zhì)子緩沖能力影響不顯著,但DNA延滯能力減弱。氮磷比(N/P)為6的CS-g-mPEG-g-PEI-x/DNA復合物能夠有效抵抗0.10μg/μL肝素鈉和2.0 U核酸酶的破壞。10%血清條件下Hep-2細胞中的基因轉(zhuǎn)運結(jié)果表明,CS-g-mPEG-g-PEI聚合物細胞毒性低,mPEG-NH2接枝率為2.9%時基因轉(zhuǎn)運效率最好。從殼寡糖出發(fā),合成了酸敏感酰腙鍵和還原敏感二硫鍵交聯(lián)雙重刺激響應(yīng)殼聚糖(SRCS):采用前述方法,對SRCS進行mPEG-NH2和PEI共價接枝修飾,得到SRCS-g-mPEG-g-PEI聚合物。利用1HNMR、13C-CP/MAS NMR、FTIR、UV和GPC等手段對產(chǎn)物結(jié)構(gòu)進行了表征。SRCS-g-mPEG-g-PEI能夠有效壓縮DNA,形成的復合物(N/P≥10)在0.10μg/μL肝素鈉、2.0 U核酸酶及25%血清中能夠穩(wěn)定存在,在pH=5.0的酸環(huán)境或10 mM DTT的還原環(huán)境中可以響應(yīng)釋放DNA。Hep-2細胞中的基因轉(zhuǎn)運結(jié)果表明,SRCS-g-mPEG-g-PEI在10%血清條件下具有良好的基因轉(zhuǎn)運性能,有望成為安全、高效的殼聚糖基非病毒基因載體。
[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，
本文編號：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，
本文編號：2346653
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