基于改性聚乙烯亞胺的基因藥物傳遞體系研究
發(fā)布時間：2019-04-28 11:14
【摘要】：針對目前基因及藥物傳遞體系前沿的研究方向,我們將改性聚乙烯亞胺(PEI)和超分子組裝技術(shù)結(jié)合,為構(gòu)建新型的基因傳遞體系和基因、藥物控釋涂層提供新途徑。 本研究首先成功合成了具有pH刺激響應(yīng)電荷逆轉(zhuǎn)的大分子CPEI,其在中性條件下表面電位在-20mV左右,呈負(fù)電性,但在酸性條件下,呈現(xiàn)出電荷逆轉(zhuǎn)的特點(diǎn)帶上正電,表面電位在+2.0mV。隨著在酸性環(huán)境中時間的增加,其正電性也由于側(cè)基的水解而增強(qiáng)。以此大分子為組分,與預(yù)組裝得到的PEI-DNA復(fù)合粒子進(jìn)行層層自組裝,構(gòu)建得到負(fù)載有PEI-DNA復(fù)合粒子的多層膜,12雙層的膜厚接近120nm。進(jìn)一步的研究表明,該多層膜呈現(xiàn)出了CPEI所帶來的電荷逆轉(zhuǎn)的特點(diǎn),能夠?qū)H發(fā)生刺激響應(yīng),在pH小于6.0的情況下釋放得到含有DNA的復(fù)合粒子。這一具有pH響應(yīng)功能的基因薄膜涂層為原位釋放、轉(zhuǎn)染細(xì)胞提供了新的可能。 研究進(jìn)一步制備了二茂鐵修飾的PEI (BPEI-Fc),利用二茂鐵的疏水性誘導(dǎo)形成膠束,其臨界膠束濃度為0.046mg/mL。該膠束成功負(fù)載了疏水性的小分子藥物阿霉素,包覆量為7.7%。載有阿霉素的膠束與DNA成功復(fù)合形成藥物基因雙傳遞的預(yù)組裝體,并與磺化葡聚糖進(jìn)行了層層自組裝,構(gòu)建了具有藥物基因雙傳遞功能的多層膜。該種多層膜50雙層能夠負(fù)載阿霉素6.951μg/cm2,并能在雙氧水刺激下釋放出接近90%的阿霉素。在隨后的細(xì)胞試驗(yàn)中發(fā)現(xiàn),載入多層膜內(nèi)的阿霉素確實(shí)能夠抑制肝癌細(xì)胞的生長,50雙層的多層膜72h小時相對TCPS的細(xì)胞活性不到20%。綠色熒光蛋白質(zhì)粒DNA的轉(zhuǎn)染實(shí)驗(yàn)也證實(shí),多層膜能夠?qū)崿F(xiàn)HEK293細(xì)胞和肝癌細(xì)胞的轉(zhuǎn)染和蛋白質(zhì)的表達(dá)。這一具備藥物和基因雙傳遞功能的薄膜涂層為解決癌癥抗藥性問題提供了新的途徑。 最后,利用二茂鐵與β-環(huán)糊精的主客體嵌套作用,將已合成的二茂鐵修飾的PEI,與β-環(huán)糊精充分復(fù)合形成粒徑在169nm的粒子。將粒子與DNA復(fù)合后,構(gòu)建了β-環(huán)糊精輔助傳遞DNA的液相傳遞粒子,考察了不同N/P值下的復(fù)合粒子的理化特性及復(fù)合規(guī)律。在N/P值為1時,DNA尚未完全與BPEI-Fc-CD復(fù)合,在N/P值為2時,DNA才完全被BPEI-Fc-CD復(fù)合到粒子中。當(dāng)N/P值大于等于7時,DNA才被完全的包覆在了粒子內(nèi)部。穩(wěn)定性實(shí)驗(yàn)表明,粒子在體外細(xì)胞培養(yǎng)環(huán)境中相對穩(wěn)定,沒有發(fā)生明顯的聚集。在轉(zhuǎn)染實(shí)驗(yàn)中發(fā)現(xiàn),該種液相的傳遞基因的復(fù)合粒子,在環(huán)糊精的輔助作用下,確實(shí)能夠?qū)崿F(xiàn)相對于傳統(tǒng)PEI而言更高的轉(zhuǎn)染率和更好的細(xì)胞相容性,其基因轉(zhuǎn)染效率超過18%。這一結(jié)果為構(gòu)建理想的基因傳遞體系提供了新的可能。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341
本文編號：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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R341
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 吳濤,張鳳魚;非病毒型基因傳遞系統(tǒng)[J];沈陽藥科大學(xué)學(xué)報;2001年04期
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