载基因壳聚糖纳米粒的制备及其特性的研究

目的制备壳聚糖纳米粒,并连接上质粒,研究壳聚糖纳米粒的特性及其对DNA的结合及保护能力。方法采用离子交联法制备壳聚糖纳米粒,并用喷金扫描电子显微镜检测,了解粒径的分布与形态;通过静电吸附作用连接上pGenesil-1质粒(报告基因);经琼脂糖凝胶电泳分析壳聚糖纳米载体与质粒DNA的结合能力,及不同pH值的壳聚糖纳米粒对质粒DNA的结合能力;并通过DnaseⅠ消化壳聚糖纳米-质粒结合物以观察壳聚糖纳米载体对质粒的保护作用。结果喷金扫描电镜检测证实壳聚糖纳米粒呈均匀分散的球形颗粒,平均直径为5nm;琼脂糖凝胶电泳的结果显示壳聚糖纳米粒能有效地结合载体pGenesil-1质粒;不同pH值的壳聚糖纳米粒对质粒的保护作用不同,当pH值<7时壳聚糖纳米载体能100%结合质粒;DnaseⅠ消化试验证实壳聚糖纳米载体对质粒DNA有保护作用。结论采用离子交联法制备出粒径较小、均匀的壳聚糖纳米粒,并且壳聚糖纳米粒能有效地连接上质粒并对其有保护作用。

Preparation and characteristics of chitosan nanoparticles as a gene vector

Objective To prepare the plasmid-binding chitosan nanoparticle （CS-NP） and explore its biologic properties, DNA affinity and protection. Methods Ion cross-linkage was used to prepare CS-NP, and the diametric distribution and morphology of CS-NP were tested by gold-labeling scanning electron microscopy （SEM）. The binding of pGenesil-1 （reporter gene） to CS-NP was achieved by static electric attraction. The affinity and protective effect of CS-NP for plasmid under different pH conditions was analyzed with agarose gel electrophoresis. Furthermore, DNA- protecting capacity of CS-NP complex was confirmed with Dnase I digestion. Results Under gold-labeling SEM, CS-NP were round, even-distributed nanoparticles measuring 5 nm in diameter. Agarose gel electrophoresis showed effective uptake of pGenesil-1 by CS-NP, while the protective action of CS-NP on plasmid varied with different pH conditions. At pH 〈7, nearly 100% of plasmids were CS-NP-bound. Protective action of CS-NP on plasmid was confirmed by Dnase Ⅰ . Conclusion CS-NP prepared by ion cross-linkage appeared as tiny, even-distributed particles that can effectively binds to plasmids to protect DNA.