【摘要】：多功能脂質(zhì)體作為基因載體,在基因治療中的應(yīng)用越來越受到重視,展現(xiàn)出了廣闊的應(yīng)用前景。本文以葉酸-聚乙二醇-膽固醇半琥珀酸酯(F-PEG-CHEMS)和硬脂酰八聚精氨酸(R8)作為功能成分,構(gòu)建多功能脂質(zhì)體,并把它作為ASODN和質(zhì)粒DNA的載體,考察其理化性質(zhì)和細(xì)胞的攝取行為與轉(zhuǎn)染效果,探索其作為基因藥物細(xì)胞內(nèi)給藥系統(tǒng)的可行性。 首先,本文在調(diào)研有關(guān)文獻(xiàn)基礎(chǔ)上,設(shè)計(jì)出了膽固醇半琥珀酸酯(CHEMS)、聚乙二醇單甲醚-膽固醇半琥珀酸酯(MPEG-CHEMS)和F-PEG-CHEMS的合成路線。CHEMS是以膽固醇和丁二酸酐為原料通過酯化反應(yīng)制備；MPEG-CHEMS是以聚乙二醇單甲醚(MPEG)和CHEMS為原料通過酯化反應(yīng)制備；F-PEG-CHEMS的合成分三步完成：首先葉酸與聚乙二醇二胺(NH2-PEG-NH2)反應(yīng)制備F-PEG2000-NH2,然后膽固醇半琥珀酸酯(CHEMS)與N-羥基琥珀酰亞胺(NHS)反應(yīng)制備NHS-CHEMS,最后F-PEG-NH2與NHS-CHEMS反應(yīng),得到F-PEG-CHEMS。合成并純化的產(chǎn)物經(jīng)TLC, IR,.1H-HMR質(zhì)譜和1H-HMR譜分析,確證為目標(biāo)產(chǎn)物。合成并純化了的脂質(zhì)體膜材MPEG-CHEMS與F-PEG-CHEMS,為多功能脂質(zhì)體的制備奠定了基礎(chǔ)。 其次,用薄膜分散法制備普通脂質(zhì)體,并把R8水溶液與普通脂質(zhì)體在45℃孵育1h,制備R8修飾的脂質(zhì)體(R8-Lip),同時對其進(jìn)行理化性質(zhì)和細(xì)胞水平的評價(jià)。實(shí)驗(yàn)結(jié)果表明,普通脂質(zhì)體經(jīng)R8修飾后,粒徑和分布基本不變,而zeta電位由-10.24mV變?yōu)?38.69mV。當(dāng)R8-Lip/ASODN復(fù)合物中+／-超過4：1時,ASODN已與R8-Lip完全結(jié)合成帶正電的復(fù)合物；當(dāng)R8-Lip中R8的濃度小于10μg/mL時,細(xì)胞存活率大于75%;R8-Lip對ASODN細(xì)胞攝取的促進(jìn)作用與R8的濃度有關(guān),當(dāng)R8的質(zhì)量濃度由0.3μg/mL增加到9.6μg/mL(+／-由1：2增加到16：1),細(xì)胞內(nèi)熒光強(qiáng)度逐漸增加；當(dāng)R8-Lip/ASODN復(fù)合物中+／-=16：1時,R8對ASODN攝取的促進(jìn)作用強(qiáng)于市售的轉(zhuǎn)染試劑Lipofectamine2000。 在R8-Lip表面PEG化修飾后,促進(jìn)細(xì)胞攝取ASODN的效果很差。為了進(jìn)一步提高細(xì)胞攝取效果,在PEG末端接上葉酸配體,構(gòu)建了多功能脂質(zhì)體R8-Lip-F,并對其進(jìn)行理化性質(zhì)和細(xì)胞水平的評價(jià)。實(shí)驗(yàn)結(jié)果表明,該脂質(zhì)體的形態(tài)規(guī)整,幾乎全部呈球形。R8-Lip-F/ASODN在+／-為16：1時復(fù)合物完全被阻滯在上樣口。R8-Lip-F/ASODN復(fù)合物在6h內(nèi)的細(xì)胞攝取量和陽性率隨時間的增加而增加。R8-Lip-F/ASODN復(fù)合物通過內(nèi)吞途徑入胞,且具有能量依賴性。其入胞機(jī)制主要是網(wǎng)格蛋白介導(dǎo)的內(nèi)吞和巨胞飲作用,并且細(xì)胞攝取量會因葉酸的競爭性抑制而降低。ASODN入胞后大部分分布在胞漿中,細(xì)胞核中幾乎沒有。與市售的轉(zhuǎn)染試劑Lipofectamine2000相比,R8-Lip-F對ASODN攝取的促進(jìn)作用要強(qiáng)一些,但其對質(zhì)粒DNA的轉(zhuǎn)染幾乎無促進(jìn)作用,轉(zhuǎn)染效果遠(yuǎn)差于市售的轉(zhuǎn)染試劑Lipofectamine2000。 最后,以DOTAP脂質(zhì)體為基礎(chǔ),并在其中引入F-PEG-CHEMS與R8,構(gòu)建多功能陽離子脂質(zhì)體R8-DOTAP-F,同時對其進(jìn)行理化性質(zhì)和細(xì)胞水平的評價(jià)。實(shí)驗(yàn)結(jié)果表明,R8-DOTAP-F的形態(tài)規(guī)整,幾乎全部呈球形。在R8-DOTAP-F復(fù)合物中DOTAP與pDNA或ASODN質(zhì)量比為30(正負(fù)電荷比或N/P約為13：1)時,pDNA和ASODN被結(jié)合完全。R8-DOTAP-F/ASODN復(fù)合物在6h內(nèi)的細(xì)胞攝取量和陽性率隨時間的增加而增加。對脂質(zhì)體進(jìn)行葉酸配體修飾,可以明顯提高脂質(zhì)體復(fù)合物的細(xì)胞攝取與轉(zhuǎn)染效果,并且,對脂質(zhì)體進(jìn)行R8修飾可以進(jìn)一步提高細(xì)胞轉(zhuǎn)染與攝取效果。R8-Lip-F/ASODN復(fù)合物通過內(nèi)吞途徑入胞,且具有能量依賴性。其入胞機(jī)制主要是網(wǎng)格蛋白介導(dǎo)的內(nèi)吞和巨胞飲作用,并且細(xì)胞攝取量會因葉酸的競爭性抑制而降低。入胞后ASODN大部分分布在細(xì)胞核中,溶酶體中幾乎沒有。與市售的轉(zhuǎn)染試劑Lipofectamine2000相比,R8-DOTAP-F對ASODN攝取的促進(jìn)作用要強(qiáng)很多,但其對質(zhì)粒DNA轉(zhuǎn)染的促進(jìn)作用要略差一些。
[Abstract]:The multifunctional liposome is used as a gene vector, and the application of the multifunctional liposome in gene therapy is more and more important, and has a wide application prospect. A multifunctional liposome was constructed by taking folic acid-polyethylene glycol-cholesterol semi-succinate (F-PEG-CHEMS) and stearin 8-poly arginine (R8) as functional components, and taking it as carrier of ASODN and plasmid DNA. To explore the feasibility of the drug delivery system as a gene drug cell. Firstly, the synthesis of cholesterol semi-succinate (CHEMS), polyethylene glycol monomethyl ether-cholesterol semi-succinate (MPEG-CHEMS) and F-PEG-CHEMS was designed based on the literature. The synthesis of F-PEG-CHEMS is carried out by reaction of folic acid with polyethylene glycol diamine (NH2-PEG-NH2). H2 and then reacting cholesterol semi-succinic acid ester (CHEMS) with N-hydroxyamber to prepare the F-CHEMS, and finally reacting the F-PEG-NH2 with the NHS-CHEMS to obtain the F-PEG-NH2. MS. The synthesized and purified products were identified as targets by TLC, IR,. 1H-HMR mass spectrometry and 1H-HMR spectroscopy The synthesized and purified liposome membrane material MPEG-CHEMS and F-PEG-CHEMS have laid the foundation for the preparation of multifunctional liposomes. secondly, preparing the common liposome by a thin film dispersion method and incubating the R8 aqueous solution with the common liposome at 45 DEG C for 1h to prepare R8-modified liposome (R8-Lip), The experimental results show that the particle size and distribution of the liposomes are basically unchanged after R8 is modified, and the zeta potential is changed from -10. 24mV to + 38.. 69mV. When +/-exceeds 4: 1 in R8-Lip/ ASODN complex, ASODN has been fully integrated with R8-Lip to positively charged complex; when R8-Lip is less than 10 & mu; g/ mL, cell survival is greater than 75%; R8-Lip contributes to the uptake of ASODN cells and R8 The concentration of R8 is related to the increase of fluorescence intensity in cells when the mass concentration of R8 is increased from 0.3. m u.g/ mL to 9. 6. m u.g/ mL (+/-increased from 1: 2 to 16: 1); when +/-= 16: 1 in R8-Lip/ ASODN complex, R8 promotes the uptake of ASODN stronger than the commercially available transfection reagent, Lipofectin e2000. After PEGylation modification on R8-Lip surface, promote cell uptake AS In order to further improve the effect of cell uptake, a multifunctional liposome R8-Lip-F was constructed and its physicochemical properties were studied in order to further improve the effect of cell uptake. The results show that the morphology of the liposome is regular. Almost all spherical. R8-Lip-F/ ASODN is completed at +/-16: 1 The cellular uptake and positive rate of the R8-Lip-F/ ASODN complex in 6h increased with the increase in time increases. R8-Lip-F/ ASODN complexes are introduced into the cell by an endocytic pathway, It has an energy-dependent mechanism. Its intracellular mechanism is mainly protein-mediated endocytic and giant cell drinking, and the cellular uptake is due to folic acid. competitive inhibition decreased. The majority of ASODN was distributed in cytoplasm after ASODN was introduced into the cytoplasm. The effect of R8-Lip-F on the uptake of ASODN was lower than that of the transfection reagent which was sold in the city. However, it had little effect on the transfection of plasmid DNA. The transfection efficiency was far from the transfection reagent of the market. mine2000, finally, using DOTAP liposome as the basis, introducing F-PEG-CHEMS and R8 in it, constructing multifunctional cationic liposome R8-DOTAP-F, The results show that R8-DOTAP-F In the R8-DOTAP-F complex, pDNA is present in the R8-DOTAP-F complex with a mass ratio of DOTAP to pDNA or ASODN of 30 (positive/ negative charge ratio or N/ P of about 13: 1). and ASODN are combined completely. R8-DOTAP-F/ ASODN complex has cell intake and yang in 6h According to the modification of the folic acid ligand on the liposome, the cell uptake and transfection effect of the liposome complex can be obviously improved, and the R8 modification on the liposome can further Enhancement of cell transfection and uptake results. R8-Lip-F/ ASODN complexes pass endocytic pathways The intracellular mechanism is primarily a protein-mediated endocytic and macrocellular uptake, and the cellular uptake is due to The competitive inhibition of folic acid decreases. The majority of ASODN after entering the cell is distributed in cells. Almost none of the lysosomes in the nucleus. Compared with the transfection reagent which was sold in the market, R8-DOTAP-F promoted the uptake of ASODN.
【學(xué)位授予單位】：浙江工業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R943

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