Wang Wei, Tang Ning, Zhang Chun-ling, Liu Xing-jun, Hu Han, Zhang Zhi-xiang, Liang Wei
National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Yao Xue Xue Bao. 2006 Feb;41(2):142-8.
To prepare the PEGylated liposomes modified with cell penetrating peptides, which protect siRNA from nuclease degradation and deliver efficiently siRNA into cells to facilitate silencing of target gene.
The purity of R8-PEG-PE and pNP-PEG-PE was detected by HPLC; the quantity of R8, PEG-DPPE modified R8, and R8 attached to the out membrane surface of the liposomal siRNA by transfer from R8-PEG-DPPE micelles to the liposomes was tested by fluorescence; Size and size distribution of siRNA loaded liposomes with and without attached R8 were determined by Zetasizer 5000; A comparison of mediated siRNA transfection efficiency between R8-liposomes and lipofectamine 2000 was examined by individual inside cell fluorescence intensity; The growth inhibition of small cell lung carcinoma NCI-H446 cells treated with R8-liposomal hdm2-siRNA or lipofectamine 2000-hdm2-siRNA complex was tested by MTT assay.
The retention times of PEG-DPPE and R8-PEG-DPPE were 9.0 min and 7.8 min, respectively. Fluorescence scanning indicated that lipids composed of liposomes and siRNAs didn't interfere to the determination of R8 when it was attached to the liposomal siRNA. The cells treated with R8-liposomal hdm2-siRNA significantly enhanced the cellular uptake of hdm2-siRNA and facilitated the functions of hdm2-siRNA through silencing of target gene which, in turn, inhibited tumor cell growth, compared with lipofectamine 2000.
The R8 attached liposomes are shown to be powerful carriers for delivery siRNAs into cell to silence targeted gene.
制备用细胞穿透肽修饰的聚乙二醇化脂质体，其可保护小干扰RNA（siRNA）不被核酸酶降解，并将siRNA高效递送至细胞内以促进靶基因沉默。
采用高效液相色谱法检测R8-聚乙二醇-磷脂酰乙醇胺（R8-PEG-PE）和对硝基苯-聚乙二醇-磷脂酰乙醇胺（pNP-PEG-PE）的纯度；通过荧光检测从R8-PEG-DPPE胶束转移至脂质体后，脂质体表面结合的R8、聚乙二醇-二棕榈酰磷脂酰乙醇胺（PEG-DPPE）修饰的R8以及与脂质体结合的R8的量；用Zetasizer 5000测定负载有或未结合R8的siRNA脂质体的大小和大小分布；通过细胞内荧光强度检测比较R8-脂质体和脂质体转染试剂2000介导的siRNA转染效率；采用噻唑蓝（MTT）法检测用R8-脂质体hdm2-siRNA或脂质体转染试剂2000-hdm2-siRNA复合物处理的小细胞肺癌NCI-H446细胞的生长抑制情况。
PEG-DPPE和R8-PEG-DPPE的保留时间分别为9.0分钟和7.8分钟。荧光扫描表明，脂质体和siRNA组成的脂质不干扰R8结合到脂质体siRNA上时的测定。与脂质体转染试剂2000相比，用R8-脂质体hdm2-siRNA处理的细胞显著增强了hdm2-siRNA的细胞摄取，并通过靶基因沉默促进了hdm2-siRNA的功能，进而抑制肿瘤细胞生长。
结合R8的脂质体被证明是将siRNA递送至细胞内以使靶向基因沉默的有效载体。
