载鱼精蛋白-pDNA复合物固体脂质纳米粒的初步研究

Preliminary Studies on Protamine-pDNA Complex Loaded Solid Lipid Nanoparticles

目的制备非病毒基因载体载鱼精蛋白-pDNA复合物的固体脂质纳米粒(PD-SLN),PD-SLN由多聚阳离子缩合DNA内核与一个脂质外壳组成,从而构成多功能信封式纳米载体(multifunctional envelope-type nano device,MEND)结构,研究其特征,对DNA的保护作用以及DNA的体外释放性质。方法分别采用溶剂扩散法和复乳法制备纳米粒;用透射电镜观察形态;用Zeta电位测定仪测定粒径、多分散指数和Zeta电位;用荧光分光光度法测定基因包封率;分别用琼脂糖凝胶电泳观察复合物及PD-SLN保护pDNA抵抗剧烈外力和核酸酶的降解情况;采用双室扩散法对PD-SLN进行体外释放研究。结果用2种方法制备的SLN呈球形和类球形,平均粒径分别为(231±13.7)和(627±22.9)nm,Zeta电位分别为(-17.8±3.2)和(-25.2±2.7)mV,包封率分别为(41.5±3.62)%和(56.5±5.28)%。pDNA保护性试验表明,PD-SLN对pDNA有保护作用。体外释放实验结果表明PD-SLN缓释能力强。结论PD-SLN是一种制备工艺简单,体外缓释能力好,对pDNA保护性强,具有一定应用前景的非病毒基因载体。

OBJECTIVE To prepare nonviral vector of solid lipid nanoparticles carrying protamine-pDNA complex （PD-SLN）, to develop a Multifunctional Envelope-type Nano Device （MEND） structure, in which the core of a plasmid DNA, condensed by a polycation, is encapsulated by a lipid envelope, then the nanoparticles＇ characteristics, the protection ability and the DNA release property in vitro were evaluated. METHODS PD-SLN was prepared by emulsion solvent diffusion method and double emulsion solvent evaporation method, respectively. The morphology of PD-SLN was observed by transmission electron microscopy. The particle sizes, polydispersity, Zeta potential were measured by nanoparticlc size analyser. Encapsulating efficiency of pDNA was determined by fluorescence spectrometer. The protection of complex and PD-SLN from intensive force and nuclease degradation were evaluate by agarose gel electrophoresis,respectively. The method of two-compartment diffusion was employed to investigate the releasing character of DNA from PD- SLN. RESULTS The morphology of PD-SLN were approximately spherical. The average particle sizes of PD-SLN obtained by the two different methods were （231 ± 13.7 ） and （627 ± 22.9） nm, respectively. The Zeta potentials were （ - 17. 8 ± 3.2） and （ - 25.2 ± 2.7 ） mV, respectively. Encapsulating efficiencies were （41.5 ± 3.62） % and （ 56. 5 ± 5.28 ） %, respectively. The intensive agitation and nuclease degradation test results confirmed that the pDNA was protected considerably. PD-SLN Maintained sustained-release of pD- NA for several days in vitro. CONCLUSION PD-SLN could be prepared easily with small particle sizes, excellent sustained-release activity, and protection of pDNA. In vitro studies have showed that PD-SLN could be a promising device,which has the potential to make in vivo cancer gene therapy achievable.