转运SiRNA的双靶向长循环多功能信封式纳米基因载体的研究

Non-viral vectors for gene delivery usually have the following characteristics, such as lower toxicity, immunogenicity, carcinogenicity, and transfection efficiency than the viral vectors. Multifunctional envelope-type nano device (MEND) as a non-viral vector, combines the features of liposome and nanoparticle, is prone to enter into cells and enhance the transfection efficiency. This study will prepare the dual targeting & long circulating MEND. The lipid material of MEND will be optimized to get a new chemical modified product glycyrrhetinic acid(GA)-PEG-Peptide(MMP cleavable peptide)-DOPE in order to prevent lysosomal degradation and enhance nuclear translocation. Commonly, the hepatic tumor formation occurs in the hepatic parenchymal cells, the hepatic non-parenchymal cells have phagocytosis to the exogenous material. Modifying with GA will enhance the positive targeting of MEND for the hepatic parenchymal cells, because of the overexpression of GA receptor on the surface of hepatic parenchymal cells. The existence of PEG will enhance the stability, prolong the plasma half-life time of MEND. PEG could obstruct the endocytosis of MEND because of its steric hindrance effects, when the MEND reaches the tumor tissue, the MMP cleavable peptide can be cleaved by MMP which is overexpressed in the tumor cells, then GA-PEG will be fragmented and the unmodified MEND will be easy for endocytosis, thereby to achieve the targeting effect for the hepatic parenchymal tumor cells. We'll determine the transfection efficiency in vitro and the stability in the plasma of SiRNA. We hope that the results of this study will be the base of further research of clarification the effects and mechanism of specifical recognizing and entering into the hepatic parenchymal tumor cells, long circulating & targeting for the hepatic parenchymal cells and the pharmacodynamic effect on the tumor cells in vitro & in vivo.

基因治疗中，非病毒基因载体具低毒性低免疫原性特点，但仍存在靶向性和转染效率较低的问题。本课题制备一种双靶向转运siRNA的多功能信封式纳米装置(MEND),对其脂质成分进行了修饰，修饰后的脂质分子结构为：甘草次酸(GA)-PEG-peptide(基质金属蛋白酶MMP底物)-二油酰磷脂酰乙醇胺(DOPE)。其中PEG能增加MEND血浆稳定性；GA对肝实质细胞具有主动靶向作用；由于PEG阻碍MEND进入细胞，而peptide可被肿瘤组织过度表达的MMP裂解，导致PEG脱落，去掉修饰的MEND易于被肿瘤细胞内吞，从而具有对肿瘤细胞的被动靶向性；考察该制剂对siRNA的体内外转染效率、血浆稳定性的影响。本课题结果将为进一步阐明SiRNA的双靶向MEND制剂的肝肿瘤细胞靶向性、转运机制、SiRNA的细胞和动物药效提供研究基础。

制备了一种转运siRNA 的双靶向长循环多功能信封式纳米基因载体（MEND），合成了基本结构GA-PEG-PEPTIDE-DOPE-脂质体（包裹SiRNA），经核磁共振和质谱进行了结构确证。其中GA为甘草次酸，可以靶向识别肝脏实质细胞；PEG为聚乙二醇，能够增加SiRNA-MEND在血液中的的稳定性，延长血浆半衰期，但其空间位阻作用会阻碍MEND进入细胞；PEPTIDE为一个肽段，是基质金属蛋白酶（MMP）的底物，由于肿瘤细胞外分泌大量MMP，因此当该MEND到达肿瘤细胞靶部位时，肽段被MMP 水解后PEG 脱落，不会影响到载体基因进入细胞，导致对肿瘤细胞的靶向性。SiRNA与聚赖氨酸（PLL）以1:20的比例进行缩合，体积减少易于被脂质体包裹，同时增加其血浆稳定性；采用薄膜水化法制备了SiRNA脂质体；经透射电镜观察，制备的MEND为比较圆整的脂质双层结构，经激光粒度电位仪测定，MEND粒径范围为163.5±20 nm，zeta电位接近零；采用透析法测定SiRNA的包封率为82.8%；测定了MMP对该MEND的酶解作用，结果显示PEPETIDE 可以成功被MMP降解导致PEG的脱落；测定了MEND中SiRNA在血清中的稳定性，结果表明，经GA-PEG-PEPTIDE修饰的MEND 包裹的SiRNA在血清中稳定性明显提高；测定了MEND对体外培养的BEL-7402细胞的细胞存活率的影响，结果表明经所有浓度的MEND作用后的BEL-7402细胞存活率均大于90%；检测了Cy3-SiRNA-MEND对BEL-7402细胞的转染效率，结果显示，经修饰的MEND包裹的Cy3-SiIRNA对BEL-7402细胞的转染效率显著高于未经修饰的Cy3-SiIRNA脂质体和裸露Cy3-SiIRNA；进行了MEND的细胞内吞实验，结果显示SiRNA-MEND能够顺利进入细胞质；测定了SiRNA-MEND对BEL-7402细胞的迁徙抑制作用，结果表明经ras-SiRNA-MEND转染的细胞的迁徙转移能力显著下降；采用实时荧光定量RT-PCR法测定了ras-SiRNA-MEND的基因沉默作用，结果表明其基因沉默率为空白MEND的78.61倍。实验结果表明该双靶向长循环多功能信封式纳米SiRNA转运载体能够高效低毒地转染肿瘤细胞，导致基因沉默。

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