还原响应型嵌段共聚物自组装纳米胶束作为siRNA运输载体的研究

摘要

采用二重氢键为引导、双二硫键为连接单元的方法合成嵌段共聚物 PEG2000-PLA3000-PEI1200-PLA3000-PEG2000,其自组装形成的纳米胶束可作为小干扰核糖核酸(siRNA)运输载体.采用核磁(1 H-NM R)、凝胶渗透色谱(GPC)、激光共聚焦显微镜(CLSM)等检测方法进行表征.结果表明:在二重氢键引导下合成嵌段共聚物,其自组装形成纳米胶束的临界胶束浓度(CM C)为0.052 mg/mL,粒径为(32 ± 0.1)nm,表面电势为(46.9 ± 0.7)mV.负载siRNA的胶束粒径为(35 ± 0.3)nm,表面电势为(27.2 ± 1.1)mV.激光共聚焦显微镜的检测证明纳米胶束可携带siRNA进入细胞.%Using double hydrogen bonds as the guide and double disulfide bond as the connecting unit,the redox-responsive amphiphilic cationic multi-block copolymer PEG2000-PLA3000-PEI1200-PLA3000-PEG2000was synthesized and self-assembled into micelles for delivery of short-interfering ribonucleic acid(siRNA).The copolymer was characterized by Nuclear Magnetic Resonance(NMR),Gel Permeation Chromatography(GPC),Confocal Laser Scanning Microscope(CLSM)and Transmission Electron Microscope(TEM). The micellar particles were observed by Dynamic Light Scattering(DLS).The size of blank micelles was (32 ± 0.1)nm and the Zeta potential was(46.9 ± 0.7)mV.The size of siRNA-loaded micelles was(35 ± 0.3)nm and the Zeta potential was(27.2 ± 1.1)mV.The micelles observed by TEM were spherical.The Critical Micelle Concentration(CMC)of self-assembled micelles was determined with 1,6-two phenyl-1,3, 5-hexene(DPH)as a UV molecular probe,which was 0.052 mg/mL and indicated that the micelles could be stable in dilute solutions.The micellar particles kept stable under the environment of 37 ℃ for 18 d,as reflected by DLS results.The micellar particles could be destructed by dithiothreitol(DTT),confirming that reductive environment could trigger gene release.M T T assay results demonstrated that the blank micelles had low cytotoxicity and good biocompatibility even the mass concentration was up to 100 μg/mL. Gel retardation assay results demonstrated that in redox environment(Glutathione,GS H),disulfide bonds in the polyplexes could be cleaved and most of siRNA could be released.The CLSM results confirmed the delivery of siRNA from the nanomicellar particles into the SGC 7901 cells.The reduction-responsive cationic copolymers provided a platform for constructing gene delivery system toward cancer therapy.