装载siRNA的纳米阳离子脂质体在小鼠体内的药代动力学研究

Pharmacokinetic study of nano-cationic liposomes loaded with siRNA in mice

目的:探讨siRNA药物的体内药代动力学评价方法,并分析脂质体作为药物载体的优势。方法:采用前期已构建的siRNA表达质粒及装载了该质粒的纳米隐形阳离子脂质体,将实验分为裸siRNA质粒组和siRNA质粒脂质体组,分别进行DNaseⅠ酶切和血清中稳定性的检测。再将20μg siRNA裸质粒及siRNA质粒脂质体通过尾静脉注射进入小鼠体内,分别在注射后0 min、5 min、15 min、30 min、1 h、2 h、4 h、12 h、24 h取血,采用实时荧光定量PCR方法(qPCR)对小鼠体内裸质粒及质粒脂质体进行定量检测,计算各时间点siRNA质粒的浓度。结果:裸质粒在DNaseⅠ中仅20 min就全部降解,质粒脂质体降解较缓慢,到24 h仍有(20.16±2.47)%的DNA残留;裸质粒在80%血清中20 min时仅残留(11.03±0.92)%,在1 h时基本全部降解,质粒脂质体在80%血清中24 h时仍残留(10.26±1.04)%,与裸质粒组相比,脂质体作为载体使siRNA表达质粒在DNaseⅠ及血清中的稳定性显著提高(P<0.05)。在小鼠体内脂质体包裹的质粒半衰期约为2 h,而裸质粒仅为6 min,差异具有统计学意义(P<0.05)。结论:利用qPCR方法可以完成siRNA脂质体的体内检测。阳离子脂质体能够保护核酸类药物进入体内,是一种高效的递送载体,同时脂质体能够提高药物的体内稳定性,延长药物的作用时间。

OBJECTIVE:To investigate pharmacokinetics siRNA drugs in vivo and to analyze advantages of liposomes as drug carriers.METHODS:A previously constructed siRNA expression plasmid was loaded into nano-invisible cationic liposomes and used for investigations:bare siRNA plasmid group and siRNA plasmid liposomes group.DNase I digestion and serum stability were evaluated.Then,20μg bare plasmid siRNA and liposome siRNA plasmid were injected into mice through their tail veins,and blood samples were collected at 0 min,5 min,15 min,30 min,1 h,2 h,4 h,12 h,and 24 h,respectively.Quantitative real-time PCR(qPCR)was used to quantitatively detect liposomes in mice,and the concentrations of siRNA plasmid at each time point was calculated.RESULTS:The bare plasmids were completely degraded in DNaseⅠwithin 20 min,and the degradation of plasmid liposomes was slow,with(20.16±2.47)%DNA remaining at 24 h.The bare plasmid only remained(11.03±0.92)%at 20 min in 80%serum,and was basically degraded at 1 h.The plasmid liposomes remained(10.26±1.04)%at 24 h in 80%serum.The stability of siRNA expression plasmid in DNaseⅠand serum was significantly improved by using liposomes as a carrier(P<0.05).The half-life of liposomal encapsulated plasmids in mice was about 2 h,while that of naked plasmids was only 6 min,and the difference was statistically significant(P<0.05).CONCLUSION:In vivo detection of siRNA liposomes can be accomplished by qPCR.Cationic liposomes can protect nucleic acid drugs inside the body,which is an efficient delivery carrier.At the same time,liposomes can improve stability of drugs in vivo and prolong the action time of drugs.