摘要
本研究旨在利用糖皮质激素(GC)与细胞质中糖皮质激素受体(GR)特异性结合后转移入细胞核的能力,合成一种具有细胞核靶向能力,低毒、高效的新型阳离子非病毒基因给药载体,并考察了其体内体外的转染效果。采用了效价(potency)和LogP评价GC结构与转染结果的关系。
利用Traut's试剂将5种糖皮质激素:倍他米松(BEX),地塞米松(DEX),甲基泼尼松龙(MPL)、泼尼松龙(PNL)和氢化可的松(HC)接枝到PEI 1800,得到了5种GC-PEIs,取代度都在1左右。
通过琼脂糖凝胶电泳,缓冲能力擦拭,DLS粒径测定和Zeta电位测定实验检测了GC-PEIs/pDNA的理化性质,五种GC-PEIs与pDNA的复合物理化性质相似。然而,体外HEK 293和HepG2两种细胞系上的细胞毒性和转染效率测定结果都证实了GC的修饰显著提高了PEI 1800的转染效率,但是五种GC之间有明显差异。针对GC的结构与转染效果的关系考察,采用了分子模拟对接程序考察了5种GC和GR的结合情况,并比较了用效价和LogP值来评价转染效果的可行性。
同时,采用激光共聚焦显微镜对载体在细胞内进行定位,发现BET-PEI基本实现了细胞核靶向的目的。小鼠体内肝门静脉注射BET-PEI包载pEGFP-N1复合物,倒置荧光显微成像和流式细胞术FCM测定都再一次证明了BET-PEI能显著提高转染效率,并超过PEI 25k的水平。
In this research,five different but similar Glucocorticoids(GC) were conjugated onto low molecular weight polyethylenimine(PEI 1800) by a two-step reaction by using the Traut's reagent.Our purpose was to create a kind of low cytotoxic and highly efficient gene delivery system which could complex with pDNA to form compact nanoparticles and efficiently translocate into the nucleus.
The GC-PEIs/pDNA complexes were prepared and their physico-chemical properties were investigated.The results showed that the complexes had similar physicochemical properties.The experiments of cytotoxicity and transfection efficiency of the GC-PEIs were also checked in cultured HEK 293 cell line and HepG2 cell line.The results showed that their cytotoxicity were lower than PEI 25k. Furthermore,DEX-PEI/pDNA and BET-PEI/pDNA polyplexes had similar or even greater gene delivery efficiency compared to PEI 25k/pDNA complex.The results of confocal microscopy examination and in vivo experiments on ICR mice confirmed these findings.
In order to study the relationships between the structure of GC substitute and transfection activity mediated by GC-PEIs,we performed molecular docking program to check the docking and binding energy between GC and GR.Furthermore,we compared two method used to analyze the relationships—by potency or LogP,and conclude that potency could be a potential index to indicate the transfection effects.
引文
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