包裹pHEV_(23)纳米壳聚糖的制备和特性

Preparation and characterization of chitosan-pHEV_(23) nanoparticles

背景:裸DNA在基因治疗中易被体内核酸酶降解。携载DNA疫苗的病毒类载体存在致癌和引发免疫应答等安全问题,且制备方法复杂。非病毒载体(尤其是阳离子-DNA聚合物)具有低毒,低免疫反应,能重复给药,易于大量制备等优点。壳聚糖是一种天然阳离子碱性聚多糖,具有良好的生物可降解性、生物相容性及生物安全性。目的:制备含质粒pHEV23的纳米壳聚糖,观察其表征及对DNA的保护作用。设计、时间及地点:对比观察实验,于2007-06/2008-05在浙江省医学科学院生物工程研究所完成。材料:壳聚糖,脱乙酰度88%,批号061220,由青岛利中甲壳质公司提供。质粒pHEV23为浙江省医学科学院生物工程研究所实验室提供。方法:通过脱水剂Na2SO4诱导的复凝聚沉淀法制备含质粒pHEV23的纳米壳聚糖。根据壳聚糖质量浓度1.4,0.7,0.4g/L,分别制备壳聚糖(高)-pHEV23、壳聚糖(中)-pHEV23、壳聚糖(低)-pHEV23。主要观察指标:激光纳米粒度分析仪测定壳聚糖-pHEV23粒径、Zeta电位;紫外分光光度计检测包封率;凝胶阻滞实验分析壳聚糖和pHEV23的聚合;DNaseⅠ的保护试验分析壳聚糖-pHEV23的抵抗核酸酶降解能力;释放实验评价壳聚糖-pHEV23的稳定性。结果:制备的壳聚糖-pHEV23粒径在170～470nm,其中壳聚糖(低)-pHEV23171.6nm,壳聚糖(中)-pHEV23387.0nm,壳聚糖(高)-pHEV23467.3nm。壳聚糖(低)-pHEV23Zeta电位为+22.9mV。包封率均>95%,其中壳聚糖(低)-pHEV2395.8%,壳聚糖(中)-pHEV2396.7%,壳聚糖(高)-pHEV2397.1%。凝胶阻滞分析表明,壳聚糖和pHEV23之间通过静电作用完全结合,纳米粒带正电荷。DNaseⅠ保护试验表明,壳聚糖-pHEV23能有效抵抗DNaseⅠ酶降解,对pHEV23有保护作用。稳定性试验表明,4℃保存60d,纳米粒仍能较稳定地包裹pHEV23。结论:壳聚糖-pHEV23能高效装载外源DNA,稳定性良好,并保护其免受核酸酶的降解。

BACKGROUND： Naked DNA is easily degraded by nuclease in a body during gene therapy. Viral vector carrying DNA vaccine has some problems in carcinogenesis and immune response initiation, with a complicated preparation method. Non-viral vector （especially cation-DNA polymer） has low toxin and low immune response, and can be repetitively administered, and is easily prepared in a large number. Chitosan, a natural cation basic polysaccharides, has good biodegradability, biocompatibility and biological safety. OBJECTIVE： To prepare chitosan nanoparticles carrying pHEV23 as gene carriers, and to study its characteristics and ability to protect DNA. DESIGN, TIME AND SETTING： The controlled experiment was performed at the Institute of Bioengineering, Zhejiang Academy of Medical Science from June 2007 to May 2008. MATERIALS： Chitosan, degree of deacetylation of 88%, batch number 061220, was obtained from Lizhong Chitin Co., Ltd. Plasmid pHEV23 was obtained from Laboratory of Institute of Bioengineering, Zhejiang Academy of Medical Science. METHODS： Chitosan nanoparticles （CS）-pHEV23 were prepared using a complex coacervation process induced by Na2SO4. According to mass concentration of chitosan （1.4, 0.7, 0.4 g/L）, chitosan （high）-pHEV23, chitosan （middle）-pHEV23 and chitosan （low）-pHEV23 were prepared. MAIN OUTCOME MEASURES： Chitosan-pHEV23 particle diameter and Zeta potential were determined using laser nanoparticle analyzer. Efficiency of the encapsulation was measured with a spectrophotometer. The combination manner of CS with pHEV23 was analyzed by gel retardation test. The ability t,o protect pHEV23 from nuclease degradation were evaluated using DNase Ⅰ protection test. Chitosan-pHEV23 stability was assessed by release test. RESULTS： The particle diameter of the nanoparticles were from 170 nm to 470 nm （Chitosan（low）-pHEV23 171.6 nm, Chitosan （middle）-pHEV23 387.0 nm, Chitosan （high）-pHEV23 467.3 nm）. Zeta potential（Chitosan （low）-pHEV23） ＋22.9 mV; the efficiency of the encapsulation were over 95% （Chitosan （low）-pHEV23 95.8%, Chitosan （middle）-pHEV23 96.7%, Chitosan （high）-pHEV23 97.1%）. The gel retardation assay showed that chitosan could completely combine with pHEV23 by electrostatic effect, with positive charge. The DNase Ⅰ protection test confirmed that pHEV23 could be protected from DNase Ⅰ degradation. The stability test confirmed that at 4 ℃ the pHEV23 could be parcelled well for 60 days. CONCLUSION： Chitosan-pHEV23 can effectively protect DNA from DNase Ⅰ degradation, with a good stability.