本文選題：共聚物 + 電荷翻轉(zhuǎn)��； 參考：《長(zhǎng)春理工大學(xué)》2014年碩士論文

【摘要】：本文以超支化聚乙烯亞胺(PEI)引發(fā)賴氨酸-N-內(nèi)羧酸酐(Lys-NCA)和谷氨酸-N-內(nèi)羧酸酐(Glu-NCA)單體開(kāi)環(huán)聚合,得到具有pH響應(yīng)的電荷翻轉(zhuǎn)功能的聚乙烯亞胺-聚賴氨酸-聚谷氨酸無(wú)規(guī)共聚物(PELG),該共聚物可以作為納米載體的遮蔽體系。利用烏頭酸酐(CA)對(duì)抗腫瘤藥物阿霉素(DOX)進(jìn)行修飾,得到酸敏感的小分子藥物烏頭酸酐-阿霉素(CAD)。通過(guò)核磁、凝膠滲透色譜、紅外光譜、質(zhì)譜對(duì)目標(biāo)產(chǎn)物進(jìn)行了結(jié)構(gòu)和分子量等一系列表征,證實(shí)了目標(biāo)產(chǎn)物的成功合成。 通過(guò)靜電吸附制備了PELG/PEI/CAD載藥復(fù)合物體系。Zeta電位測(cè)試證實(shí)了體系具有pH響應(yīng)的電荷翻轉(zhuǎn)功能。藥物釋放實(shí)驗(yàn)證實(shí)體系具有酸敏感的藥物釋放性能。細(xì)胞毒性實(shí)驗(yàn)證實(shí)載體材料的生物安全性良好,且PELG/PEI/CAD對(duì)HeLa細(xì)胞具有非常高的殺傷力。細(xì)胞內(nèi)吞實(shí)驗(yàn)和共聚焦照片結(jié)果顯示,體系在pH6.8(類似腫瘤微酸環(huán)境)時(shí)能夠被腫瘤細(xì)胞高效內(nèi)吞,大量的藥物可被載體攜帶進(jìn)入細(xì)胞并分布于細(xì)胞質(zhì)及細(xì)胞核中。 由于早期的工作已經(jīng)驗(yàn)證了體系的載基因性能,所以本文中進(jìn)一步主要研究基因和藥物共傳遞體系PELG/PEI/(DNA+CAD)。Zeta電位測(cè)試證實(shí)了共傳遞體系具有pH響應(yīng)的電荷翻轉(zhuǎn)功能。DNA凝膠阻滯實(shí)驗(yàn)證明體系具有良好的DNA結(jié)合能力。藥物釋放實(shí)驗(yàn)證實(shí)該共傳遞體系具有酸敏感的藥物釋放功能。利用PELG/PEI/DNA對(duì)HepG2細(xì)胞進(jìn)行DNA轉(zhuǎn)染,體系展現(xiàn)出良好的細(xì)胞轉(zhuǎn)染效率,并確定了最佳轉(zhuǎn)染質(zhì)量比：PELG/PEI/(DNA+CAD)共傳遞體系中,載入不同濃度的CAD對(duì)DNA的轉(zhuǎn)染影響并不大,并確定了體系擔(dān)載DNA和CAD的質(zhì)量比。進(jìn)一步利用p53基因作為治療基因,制備PELG/PEI/(p53+CAD)基因和藥物共傳遞體系。細(xì)胞毒性實(shí)驗(yàn)顯示共傳遞體系的半數(shù)抑制濃度(IC50)均低于單獨(dú)的載基因或載藥體系,即共傳遞體系對(duì)腫瘤細(xì)胞的殺傷力最大。激光共聚焦照片證實(shí)基因和藥物能被載體共同攜帶到腫瘤細(xì)胞中,且在pH6.8條件下,腫瘤細(xì)胞對(duì)體系復(fù)合物的內(nèi)吞效率更高,細(xì)胞內(nèi)定位的基因和藥量更多。RT-PCR實(shí)驗(yàn)證實(shí)經(jīng)過(guò)共載體系作用后的腫瘤細(xì)胞中抑癌基因p53的表達(dá)量顯著增大。凋亡實(shí)驗(yàn)顯示共載體系可使腫瘤細(xì)胞發(fā)生嚴(yán)重的凋亡,24h時(shí)可使80%的細(xì)胞凋亡,48h時(shí)癌細(xì)胞凋亡達(dá)到96%。該共傳遞體系在抗腫瘤治療中將具有很高的潛力。
[Abstract]:The ring-opening polymerization of Lys-NCAand Glu-NCA-Glu-NCA) was initiated by hyperbranched polyimide (PEI). The polyethyleneimine-poly-lysine-poly (glutamic acid) random copolymer (PELGN) with charge flipping function in response to pH was obtained. The copolymer can be used as a shelter system for nanometer carriers. Aconitine anhydride (CAA) was used to modify adriamycin (DOX) to obtain aconitine-adriamycin (CAD), a small aconitine sensitive drug. The structure and molecular weight of the target products were characterized by NMR, gel permeation chromatography, infrared spectroscopy and mass spectrometry. The PELG- / PEI- / CAD drug-carrying complex system 路Zeta potential was prepared by electrostatic adsorption. The results showed that the system had the function of charge flipping in pH response. Drug release experiments confirmed that the system has acid-sensitive drug release performance. Cytotoxicity test showed that the carrier material had good biological safety, and PELG- / PEI / CAD had a very high bactericidal effect on HeLa cells. The results of endocytosis test and confocal photography showed that the system could be efficiently ingested by tumor cells at pH 6.8 (similar to the acid-like environment of tumor). A large number of drugs can be carried into cells by vectors and distributed in the cytoplasm and nucleus. Therefore, the further study of gene and drug codelivery system PELGR / PEI / P CADN. Zeta potential test confirmed that the co-transfer system has the charge flipping function of pH response. DNA gel block test proved that the system has good DNA binding ability. The drug release assay confirmed that the co-delivery system had acid-sensitive drug release function. Using PELG- PEI / DNA to transfect HepG2 cells, the system showed good transfection efficiency, and the best transfection quality was determined to be better than that in the cotransfer system of% PELG- PEI / DNA CAD, and the effect of different concentrations of CAD on DNA transfection was not significant. The mass ratio of DNA to CAD was determined. Furthermore, using p53 gene as therapeutic gene, PELGR / PEI / p53CAD gene and drug codelivery system were prepared. The cytotoxicity test showed that the IC50 of the co-delivery system was lower than that of the single gene carrier or drug carrier system, that is, the co-delivery system had the greatest cytotoxicity to tumor cells. Laser confocal imaging showed that genes and drugs could be carried into tumor cells by vector, and at pH 6.8, tumor cells had higher endocytosis efficiency to systemic complexes. RT-PCR results showed that the expression of tumor suppressor gene p53 in tumor cells was significantly increased after co-loading. Apoptosis assay showed that cocarrier system could induce severe apoptosis in tumor cells at 24 h, and the apoptosis rate of cancer cells reached 96% at 48 h after 80% apoptosis. The co-delivery system will have high potential in anti-tumor therapy.
【學(xué)位授予單位】：長(zhǎng)春理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R943

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本文編號(hào)：2005795