载基因壳聚糖-聚乙二醇纳米粒的制备和体外评价

Preparation and in vitro evaluation of CS-PEG nanoparticles carrying gene

目的制备壳聚糖-聚乙二醇(CS-PEG)载基因纳米粒,并对其体外的相关性质进行初步研究。方法用接枝共聚法制备壳聚糖-聚乙二醇纳米粒;用复凝聚法制备载基因纳米粒;通过其形态、粒径、ζ电位、载药量、包封率和基因保护实验考察其理化特性以及基因转染效果;逆转录多聚酶链反应(RT-PCR)法和Western blot法检测转染Mcl-1 siRNA质粒后肝癌细胞中Mcl-1的表达。结果 CS-PEG纳米粒粒径为(68.9±12.3)nm,ζ电位为(32.0±6.4)mV;载基因纳米粒粒径为(111.4±16.9)nm,ζ电位为(7.5±6.4)mV,包封率为(86.8±9.7)%,载药量为(31.2±5.3)%,对基因有较好的保护作用;载基因纳米粒的最大转染效率为转染后72 h的(81.39±3.57)%,强于脂质体组且持续作用时间长(P<0.05);对肝癌细胞中Mcl-1的表达明显抑制。结论制备出低细胞毒性的CS-PEG纳米载体,载基因后粒径小,带正电荷,有很好的基因保护功能、较高的包封率和载药量,能高效率转染至细胞并有效抑制肝癌细胞中Mcl-1的表达,降低癌细胞的生存能力。

【Objective】 To prepare chitosan-polyethylene glycol nanoparticles（CS-PEG） carrying gene and study its characteristics in vitro.【Methods】 CS-PEG was synthesized through stem grafting and copolymerizing.The nanoparticles carrying gene were prepared by complex coacervation.The shape,size,zeta electric potential,envelopment rate and carry gene rate were inspected to evaluate characteristics of the vector and effect of gene transfection was detected.The expression of Mcl-1 in human hepatocellular carcinoma cells which transfected with Mcl-1 siRNA was detected by semi-quantitive reverse transcription-polymerase chain reaction（RT-PER） and Western blot.【Results】 The CS-PEG nanoparticle size was（68.9±12.3）nm and its zeta electric potential was（32.0±6.4）mV.The size,zeta electric potential,envelopment rate and carry gene rate of gene nanoparticle compound were（111.4±16.9）nm,（7.5±6.4）mV,（86.8±9.7）% and（31.2±5.3）% respectively.It also had very good protection to gene.The highest transfection rate of gene nanoparticle compound was（81.39±3.57）%（after 72h transfection）,and its persistence time after transfection was longer than that of liposome（P 0.05）.The Mcl-1 expression was suppressed after transfection in hepatocellular carcinoma cells significantly.【Conclusions】 We prepare CS-PEG nanoparticles with a low cellular toxicity.The nanoparticles have contained Mcl-1 siRNA plasmid with a small diameter,a positive charge,a good genetic protection,high encapsulation efficiency and drug loading.The compound can be transfected into hepatocellular carcinoma cells effectively,inhibit Mcl-1 expression and reduce survival ability of cancer cells.