联合包载阿霉素和siRNA阳离子脂质体的制备及体外评价

Preparation and in vitro evaluation of cationic liposomes for co-delivery of doxorubicin and siRNA

目的制备联合包载阿霉素和siRNA阳离子脂质体并进行体外评价。方法采用薄膜分散法制备载阿霉素阳离子脂质体(doxorubicin cationic liposomes,DCL),与siRNA静电复合得联合载药阳离子脂质体(liposome complexes,lipoplexes);动态激光散射法测定lipoplexes的粒径和zeta电位;透射电镜观察lipoplexes形态;超滤离心法测定lipoplexes中阿霉素和siRNA的包封率;琼脂糖凝胶阻滞实验考察lipoplexes中siRNA的结合能力;荧光显微镜观察MCF-7/ADR细胞对siRNA的摄取情况。结果所制备的lipoplexes外形圆整、分散均匀,当(2,3-二油氧基丙基)三甲基氯化铵与siRNA质量比为20时,其平均粒径为(125.7±2.7)nm,zeta电位为((45.8±1.5)m V,阿霉素包封率为(52.3±2.6)%,siRNA包封率为(88.1±1.8)%,且lipoplexes中siRNA可以紧密结合。与游离siRNA相比,lipoplexes可显著增加MCF-7/ADR细胞对siRNA的摄取。结论联合包载阿霉素和siRNA阳离子脂质体体外性质良好,能增加MCF-7/ADR细胞对siRNA的摄取,可用于小分子化疗药物和siRNA的共输送。

Objective To prepare and in vitro evaluate cationic liposomes entrapping both doxorubicin （DOX） and small interference RNA（siRNA）. Methods Thin film dispersion method was applied to prepare DOX loaded cationic liposomes, which were further bound with siRNA through electrostatic interaction to form cationic liposomes entrapping both DOX and siRNA （liposome complexes, lipoplexes）. Dynamic light scattering method was used to determine the particle size and zeta potential of lipoplexes. The morphology of lipoplexes was studied using transmission electron microscope. The entrapment efficiency of DOX and siRNA in lipoplexes were determined by centrifugal ultrafiltration. The binding ability of siRNA in lipoplexes was evaluated by agarose gel retardation assay. Fluorescence microscope was employed to study the cellular uptake of siRNA by MCF-7/ADR cells. Results The lipoplexes with round shape and uniform dispersity had a diameter of（ 141 ±2.7 ） nm, a zeta potential of（ 33.8 ± 1.5 ） mV, and an entrapment efficiency of DOX and siRNA of（52.3 ±2.6） % and（88.1 ± 1.8） % ,respectively. When DOTAP/siRNA weight ratio was up to 5, lipoplexes exhibited strong binding capacity with siRNA. Compared with free siRNA, lipoplexes could significantly enhance cellular uptake of siRNA in MCF-7/ADR cells. Conclusions Cationic liposomes entrapping both DOX and siRNA with favorable in vitro properties are demonstrated to successfully enhance cellular uptake of siRNA and can be used for co-delivery of small molecule anticancer drugs and siRNA.