壳聚糖纳米粒作为基因载体的研究:制备,特征和对DNA的保护

Chitosan Nanoparticles as Gene Vector for Mucosa Vaccination:Preparation,Characterization and Ability to Protect Plasmid DNA

目的:制备载基因壳聚糖纳米粒,研究纳米粒药剂学特征以及对DNA的保护作用.方法:复凝聚法制备纳米粒,对纳米粒的形态、粒径及分布、Zeta电位、包封率、载药量和处方影响因素进行了考察,凝胶阻滞法分析壳聚糖和pDNA的聚合方式,pDNA保护性试验考察壳聚糖纳米粒抵抗核酸酶的能力.结果:制备的pDNA/壳聚糖纳米粒为结构较紧密的不规则球形,平均粒径为(240.4±13.2)nm,多分散指数为(0.173±0.05),Zeta电位为(18.4±0.6)mV,包封率为(95.2±1.9)%,载药量为(30.7±0.8)%.凝胶阻滞分析结果表明,纳米粒荷正电,pDNA与壳聚糖之间通过静电作用而完全结合.纳米粒的粒径、Zeta电位受处方中的壳聚糖相对分子质量、N/P(壳聚糖中伯胺基数目/pDNA中磷酸基数目)比、pDNA浓度、Na2SO4浓度和pH值等因素影响.pDNA保护性试验表明,壳聚糖纳米粒对pDNA有保护作用.结论:壳聚糖可以有效凝聚pDNA,采用复凝聚法可制得200～500 nm范围荷正电的纳米粒,有较高的包封率和载药量,可有效保护pDNA免受核酸酶降解.壳聚糖作为黏膜给药的非病毒基因载体具有应用价值.

AIM： To prepare chitosan nanoparticles carrying gene vaccine and to study the nanoparticles＇ characteristics and ability to protect DNA. METHODS： pDNA/chitosan nanoparticles were prepared using a complex coacervation process. The characteristics of the nanoparticles including the morphology, particle size and its distribution, Zeta potential, association efficiency, pDNA loading and effects of the formulation variables on characterization of nanoparticles were investigated. The combination manner of chitosan with pDNA as well as ability to protect pDNA from nuclease degradation were evaluated. RESULTS： The morphology of the pDNA/chitosan nanoparticles was approximately spherical. The pharmaceutical properties of pDNA/chitosan nanoparticles were characterized by mean particle size of （240.4±13.2） nm, polydispesity index of 0.173±0.05, Zeta potential of （18.4±0.6） mV, association efficiency of （95.2±1.9） % and pDNA loading of （30.7±0.8） %, respectively. The gel retardation assay showed that chitosan could completely combine with pDNA by electrostatic effect. The particle size and Zeta potential of the nanoparticles were affected by the formulation variables such as molecular weight of chitosan, N/P ratio, pDNA concentration, coacervation agent concentration and pH. The nuclease degradation test confirmed that the pDNA could be protecte from DNase Ⅰ degradation. CONCLUSION： Chitosan can condense pDNA effectively and protected pDNA from DNase Ⅰ degradation, pDNA/chitosan nanoparticles in the particle size range of 200-500 nm with positive charge can be formulated by the coacervation process. In vitro studies have showed that chitosan could be a suitable non-viral gene vector in mucosa vaccination.