CONTRIBUTION OF CHOLESTEROL MOIETIES ATTACHED ON MPEG-b-PCL-b-PLL TO THE CELL UPTAKE, ENDOSOMAL ESCAPE AND GENE KNOCKDOWN OF THE MICELLEPLEXES OF siRNA  

作　　者：Ruo-gu Qi Su-hong Wu Yu Wang Jie Chen Zhi-gang Xie Yu-bin Huang 景遐斌 

机构地区：[1]State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences [2]University of Chinese Academy of Sciences

出　　处：《Chinese Journal of Polymer Science》2013年第6期912-923,共12页高分子科学（英文版）

基　　金：supported by the National Natural Science Foundation of China (No. 21004062);"100 Talents Program" of the Chinese Academy of Sciences (No. KGCX2-YW-802);the Ministry of Science and Technology ofChina ("973 Project", No. 2009CB930102)

摘　　要：To further enhance the transfection efficiency of a micelleplex system based on monomethoxy poly（ethylene glycol）-block-poly（e-caprolactone）-block-poly（L-lysine） （MPEG-b-PCL-b-PLL）, cholesterol （Chol） moieties are attached to the e-termini of PLL segments to obtain MPEG-b-PCL-b-PLL/Chol. The structure and morphology of the copolymer are studied by IH-NMR, TEM and DLS （dynamic light scattering）. The cytotoxicity, cell uptake, endosomal release and mRNA knockdown are studied by MTT （3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide） assay, flow cytometry, CLSM （confocal laser scanning microscopy） and RT-PCR （real-time polymerase chain reaction）. The results show that compared to their precursor MPEG-b-PCL-b-PLL, the cholesterol-grafted copolymer shows significantly lower toxicity, more rapid cellular endocytosis and endosome escape, and consequently displays enhanced siRNA transfection efficiency even at a lower N/P ratio. These improvements are ascribed to enhanced interaction of the cholesterol moieties with both cellular membrane and endosomal membrane. Moreover, effect of the PLL block length is examined. The final conclusion is that long enough PLL segments and incorporation of proper fraction of cholesterol onto the PLL segments benefit the enhancement of siRNA transfection efficiency.To further enhance the transfection efficiency of a micelleplex system based on monomethoxy poly（ethylene glycol）-block-poly（e-caprolactone）-block-poly（L-lysine） （MPEG-b-PCL-b-PLL）, cholesterol （Chol） moieties are attached to the e-termini of PLL segments to obtain MPEG-b-PCL-b-PLL/Chol. The structure and morphology of the copolymer are studied by IH-NMR, TEM and DLS （dynamic light scattering）. The cytotoxicity, cell uptake, endosomal release and mRNA knockdown are studied by MTT （3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide） assay, flow cytometry, CLSM （confocal laser scanning microscopy） and RT-PCR （real-time polymerase chain reaction）. The results show that compared to their precursor MPEG-b-PCL-b-PLL, the cholesterol-grafted copolymer shows significantly lower toxicity, more rapid cellular endocytosis and endosome escape, and consequently displays enhanced siRNA transfection efficiency even at a lower N/P ratio. These improvements are ascribed to enhanced interaction of the cholesterol moieties with both cellular membrane and endosomal membrane. Moreover, effect of the PLL block length is examined. The final conclusion is that long enough PLL segments and incorporation of proper fraction of cholesterol onto the PLL segments benefit the enhancement of siRNA transfection efficiency.

关 键 词：Amphiphilic copolymer CHOLESTEROL Endosome escape Micelleplex siRNA delivery. 

分 类 号：R341[医药卫生—基础医学]